Nutrients 2020, 12, 974; doi:10.3390/nu12040974 www.mdpi.com/journal/nutrients

Commentary

Potential Adverse Public Health Effects Afforded by

the Ingestion of Dietary Lipid Oxidation Product

Toxins: Significance of Fried Food Sources

Martin Grootveld 1,*, Benita C. Percival 1, Justine Leenders 1 and Philippe B. Wilson 1

1 Leicester School of Pharmacy, De Montfort University, The Gateway, Leicester LE1 9BH, UK;

P11279990@my365.dmu.ac.uk B.C.P.); justine.leenders@dmu.ac.uk (J.L.);

philippe.wilson@dmu.ac.uk (P.B.W.)

* Correspondence: mgrootveld@dmu.ac.uk; Tel.: +44-(0)116-250-6443

Received: 07 October 2019; Accepted: 13 March 2020; Published: 1 April 2020

Abstract: Exposure of polyunsaturated fatty acid (PUFA)-rich culinary oils (COs) to high

temperature frying practices generates high concentrations of cytotoxic and genotoxic lipid

oxidation products (LOPs) via oxygen-fueled, recycling peroxidative bursts. These toxins, including

aldehydes and epoxy-fatty acids, readily penetrate into fried foods and hence are available for

human consumption; therefore, they may pose substantial health hazards. Although previous

reports have claimed health benefits offered by the use of PUFA-laden COs for frying purposes,

these may be erroneous in view of their failure to consider the negating adverse public health threats

presented by food-transferable LOPs therein. When absorbed from the gastrointestinal (GI) system

into the systemic circulation, such LOPs may significantly contribute to enhanced risks of chronic

non-communicable diseases (NCDs), e.g. cancer, along with cardiovascular and neurological

diseases. Herein, we provide a comprehensive rationale relating to the public health threats posed

by the dietary ingestion of LOPs in fried foods. We begin with an introduction to sequential lipid

peroxidation processes, describing the noxious effects of LOP toxins generated therefrom. We

continue to discuss GI system interactions, the metabolism and biotransformation of primary lipid

hydroperoxide LOPs and their secondary products, and the toxicological properties of these agents,

prior to providing a narrative on chemically-reactive, secondary aldehydic LOPs available for

human ingestion. In view of a range of previous studies focused on their deleterious health effects

in animal and cellular model systems, some emphasis is placed on the physiological fate of the more

prevalent and toxic α,β-unsaturated aldehydes. We conclude with a description of targeted

nutritional and interventional strategies, whilst highlighting the urgent and unmet clinical need for

nutritional and epidemiological trials probing relationships between the incidence of NCDs, and

the frequency and estimated quantities of dietary LOP intake.

Keywords: lipid oxidation products; lipid hydroperoxides; aldehyde toxins; frying oils; fried foods;

cytogenicity/gentoxicity/mutagenicity; cancer; atherosclerosis; acrolein; cooking oil fumes;

maximum human dietary intake (MHDI)

1. Introduction

An increasingly large proportion of the human population consuming Western World diets

frequently ingest oxidised/peroxidised lipids, and the possibility that regular ingestion of such agents

may be deleterious to human health has recently attracted a large amount of high-impacting research

interest and focus [1–5].

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Such lipid oxidation products (LOPs), which include cytotoxic and genotoxic aldehydes, along

with their lipid hydroperoxide precursors, epoxy-fatty acids, and many other secondary or even

tertiary LOPs [6,7], arise from the peroxidative deterioration of unsaturated fatty acids (UFAs),

particularly polyunsaturated fatty acids (PUFAs), and are commonly encountered in UFA-rich

culinary oils (COs), e.g. refined, non-genetically-engineered natural corn, sunflower or soybean oils,

when exposed to high temperature frying practices at ca. 180 °C, or when stored at ambient

temperatures for prolonged durations [8–11] (Figure 1).

(a)

(b)

Figure 1. (a) Simplified reaction scheme for the peroxidation of a linoleic acid substrate molecule

present in a culinary oil linoleoylglycerol species (H represents a hydrogen atom); the conjugated

hydroperoxydiene (CHPD) species shown is one of the cis,trans-CHPD classification. (b) Molecular

structures of aldehydes arising from the fragmentation of lipid hydroperoxides (HPMs and CHPDs).

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n-Nonanal and trans-2-decenal arise from the fragmentation of oleoylglycerol-derived HPMs; n-

hexanal, trans-2-octenal and trans,trans-deca-2,4-dienal from the fragmentation of linolenoylglycerol-

derived CHPDs; and propanal, acrolein, trans-2-pentenal and trans,trans-hepta-2,4-adienal from

linolenoylglycerol-derived CHPD fragmentation. cis,trans-Deca-2,4-dienal may arise from the

thermally-induced isomerism of its trans,trans-isomer [12].

Aldehydes act as potent toxins since they are extremely chemically-reactive [1,3,7,13]. Indeed,

they cause damage to critically important biomolecules such as DNA: since they are powerful

electrophilic alkylating agents, the -unsaturated classes of these aldehydes readily alkylate DNA

base adducts, and this generally but not exclusively serves to explain their now established

mutagenic, genotoxic and carcinogenic properties. Higher concentrations of this reactive aldehyde

are effective in potently suppressing a wide range of cellular processes, which leads to indiscriminant

cellular damage and ultimately apoptosis [14].

This Commentary paper focuses on the very wide range of potential public health threats

presented by both primary LOPs (lipid hydroperoxides) and their secondary fragmentation products

(aldehydes, etc.). Primarily, Section 2 provides an extensive comprehensive review of all possible

dietary sources of LOPs, and includes subsections focused on estimates of individual dietary

aldehyde intake, most especially the molecular nature and contents of those detectable in fried foods,

along with estimated risk assessments of their consumption by humans. Section 3 delineates the GI

system interactions, in vivo absorption, metabolism and biotransformation, toxicological properties

and potential adverse health effects of these agents respectively. Data available has provided

powerful evidence that only secondary LOPs (particularly aldehydes and epoxy-acids), and not their

primary hydroperoxide precursors, are transferred to foods during high-temperature frying

practices, and that these toxins have sufficient longevity therein [12], a factor which renders them

freely available for ingestion by human populations. Section 3 begins with a full evaluation of the

biomolecular pathways and probable physiological fates of aldehydic LOPs and their conjugated

hydroperoxydiene (CHPD) precursors; potential associations of the fractional contents of different

classes of aldehydes in fried foods and those of human blood plasma are also explored for the first

time. Since many previous investigations have focused on the potential roles of dietary LOPs and

their fried food sources as major risk factors for the induction and development of atherosclerosis

and its cardiovascular disease sequelae, and cancer, a review of these involvements and their adverse

health implications are provided in Sections 4 and 5, respectively (acrolein, crotonaldehyde and

trans,trans-2,4-decadienal as inhaled or ingested carcinogens represent special cases for

consideration). Subsequently, Section 6 discusses potential mechanisms available for the toxicities

and associated adverse health effects of dietary aldehydes, with a critical consideration of the

concentrations of these agents available in the GI system, the systemic circulation and elsewhere in

vivo. Finally, Section 7 explores targeted nutrition and potential interventional strategies for

diminishing the amounts of dietary LOPs available in the human diet, and which hopefully will

provide effective barriers to health risks posed by their ingestion; alternative ‘anti-aldehyde’

prophylactic or therapeutic strategies are also discussed. This section also considers the performance

of further, more intense research investigations to establish, optimize and validate maximum human

daily intake (MHDI) values for a full range of such dietary aldehydes, rather than relying on the very

limited data currently available. Throughout the text, reference to a series of examples of the

multicomponent analysis of secondary aldehydic LOPs in COs and foods (fried or otherwise) is made

in the Figures provided. The urgent requirement for future clinical feeding trials or epidemiological

investigations focused on explorations of relationships between the incidence and/or severity of

chronic non-communicable human diseases (NCDs), and the frequency and levels of dietary LOP

intake, is stressed in the Conclusions section. In view of the focus of this Commentary on lipid

hydroperoxides and their aldehydic chain-cleavage products, the in vivo absorption and toxicities of

dietary epoxy-fatty acids (FAs) are summarized in section S1 of the Supplementary Materials section.

2. Systematic Review of Major Points

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2.1. Lipid peroxidation process: mechanistic considerations and relative susceptibilities of acylglycerol FAs

The peroxidation of UFAs during high-temperature frying practices (ca. 180 °C) represents a

complex oxidative deterioration process involving chemically-reactive free radical species (i.e.

reactive chemical species with one or more unpaired electrons), and similarly-reactive singlet oxygen

(1O2). For PUFAs, primarily this process involves the heat- and/or light-induced loss of a hydrogen

atom (H●) from relatively weak bis-allylic-CH2- function carbon-hydrogen (C-H) bonds to generate a

resonance-stabilised pentadienyl carbon-centred radical (L●), which then reacts with atmospheric

dioxygen (O2) to form a reactive peroxyl radical (LOO●) (Figure 1(a)). Structurally, these PUFA-

specific bis-allylic-CH2- functions may be viewed as being ‘sandwiched’ between two strongly

electron-withdrawing carbon-carbon double bonds (>C=C<), and this explains the weakness of their

C-H bonds, which facilitates the abstraction of H● therefrom. Once formed, LOO● radicals can then

continue to react with another, adjacent PUFA molecule to generate an additional L● radical, the

unpaired oxygen-centred electron of the peroxyl radical being converted to a more stable lipid

hydroperoxide (LOOH) species, which for PUFAs are known as conjugated hydroperoxydienes

(CHPDs). Hence, this process is known as an autocatalytic, self-propagating chain reaction, which

unless terminated by the donation of another H● from a suitable lipid-soluble chain-breaking

antioxidant (e.g. phenolic agents such as alpha-tocopherol (α-TOH)), will continue relentlessly until

all PUFAs have been consumed. In view of its autocatalytic nature, plots of aldehydic LOP

concentrations generated against time usually appear as S-shaped curves, i.e. as sigmoidal

relationships [3,12].

Once formed, CHPDs fragment to a wide range of degradation products (secondary LOPs),

particularly at high frying temperatures, and these include extremely toxic aldehydes in particular

[1,7,8]. Further CHPD deterioration products include alcohols, ketones, oxo-acids, alkanes and

alkenes [1,7,15–17], in addition to epoxy-fatty acids such as 9,10-epoxy-12-octadecenoate, which is

also known as leukotoxin [18].

However, monounsaturated fatty acids (MUFAs), which produce corresponding

hydroperoxymonoenes (HPMs) in the same manner, are much more resistant to peroxidation than

PUFAs since they only have mono-allylic-CH2- functions, with stronger C-H bonds than those of the

bis-allylic-CH2- fucntions in PUFAs. Hence, MUFAs give rise to lower or much lower levels of both

primary and secondary LOPs when heated in this manner, with a much less broader range of

secondary aldehydic LOP classifications than those derived from PUFAs, and generally only after

prolonged exposures at standard frying temperatures [13]. For example, thermal stressing of MUFA-

rich olive oil generates much lower levels of aldehydes than those observed with PUFA-rich

sunflower or corn oils, and are predominantly limited to only trans-2-alkenals and longer-chain n-

alkanals. As expected, saturated fatty acids (SFAs) are virtually completely resistant to peroxidative

damage, even at high frying temperatures. Therefore, the order of toxic LOP production in culinary

oils is PUFAs >> MUFAs >>>>> SFAs, and hence PUFA-rich culinary oils represent the riskiest choice

for use as frying media, especially when exposed to repeated frying episodes [2,3,12]. Indeed, the

relative oxidative susceptibilities of these lipid classes are 1:100:1,200:2,500 for 18-carbon chain length

fatty acids containing 0:1:2:3 >C=C< functions respectively [8]. Moreover, the rate of fragmentation of

CHPDs or HPMs to the above series of smaller molecular LOPs also increases with increasing FA

unsaturation status, i.e. it is in the order linolenoyl- > linoleoyl- >>> oleoylglycerols [9].

2.2. Dietary sources of LOPs

An extensive review of the dietary availability of LOPs is provided in Section S2 of the

Supplementary Materials. These comprise outlines of the adverse generation and analysis of LOPs in

red meat, chicken and poultry (Section S2.1); fish products (Section S2.2); dairy products (Section

S2.3); grain products (Section S2.4); fruits and vegetables (Section S2.5); and alcoholic beverages

(Section S2.6). Moreover, a full outline of the use of aldehydes as food flavouring agents is presented

in Section S2.7, and their deleterious generation in thermoplastic food packaging materials is

summarised in Section S2.8.

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A review of estimations of dietary aldehyde intake by humans, and the essential considerations

required for such criteria, is provided in this section. Used and reused culinary frying oils, and more

especially foods which have been fried therein and therefore have uptaken variable levels of such

LOP-containing media, e.g. potato chips, chicken portions and beef patties, etc., serve as rich and

very important dietary sources of these toxins (especially aldehydes) in view of their frequent human

consumption in Western diets. Therefore, this area is covered in full detail below (Section 2.3). Section

2.4 outlines the exposure of human populations to fried food sources of dietary LOPs, along with

rational estimates of their ingestions (including sub-sections focused on acrolein and 4-hydroxy-

trans-2-nonenal/4-hydroxy-trans-2-hexenal (HNE/HHE)). Moreover, section 2.5 provides

information on the risk assessment of environmental aldehydes, with special reference to the

computation of margin of exposure (MOE) values.

Wang et. al. [19] conducted an extensive review of the availability of human exposure to

environmental aldehydes from various water, food, tobacco cigarette and ambient air sources (Table

1). Of the foods listed, dietary acrolein intake from vegetables, donuts, cheese and red wine are very

high, the latter providing as much as 3.8 g/kg of this aldehyde alone! Similarly, vinegar and coffee

are very rich sources of acetaldehyde (1.06 g/kg) and furfural (255 mg/kg) respectively, whereas anise

contains a staggeringly high content of anisaldehyde (25 g/kg).

Table 1. Sources of environmental aldehydes (adapted from [19], with permission). References for

these data are provided in the Supplementary Materials section of [19].

Source Aldehyde(s) Concentration

Water Surface water (irrigation canal) acrolein 20–200 mg/L

Non – carbonated bottled water

Formaldehyde,

acetaldehyde, nonanal,

and methyl glyoxal

1.7–57.5 mg/L

Carbonated bottled water

Formaldehyde,

acetaldehyde, nonanal,

and methyl glyoxal

3.9–197 mg/L

Ground and surface water Formaldehyde and

acetaldehyde 4.5–12 mg/L

Ozone – purified water Formaldehyde and

acetaldehyde 2–20 mg/L

Foods Fruits acrolein 10–50 mg/kg

Vegetables acrolein 10–590 mg/kg

Donuts acrolein 100–900 mg/kg

Codfish fillets acrolein 100 mg/kg

Cheese acrolein 290–1300 mg/kg

Red wine acrolein 3800 mg/kg

Vinegar acetaldehyde 1.06 gm/kg

Wheaten bread butanal 51 mg/kg

Coffee furfural 255 mg/kg

Bread propanal 31 mg/kg

banana 2–hexenal 2 mg/kg

Heated butter 2–pentenal 6 mg/kg

Heated butter 2,4–nonadienal 1.5 mg/kg

Vanilla vanillin 23 g/kg

Lime, peel oil citral 130 g/kg

Anise anisaldehyde 25 g/kg

Tangerine, peel oil 2,4–decadienal 500 mg/kg

Heated lard acrolein 109 mg/L

Sunflower oil acrolein 163 mg/L

Cigarettes Mainstream acrolein 10–140 mg/cigarette

Mainstream crotonaldehyde 18.5 mg/cigarette

Mainstream acetaldehyde 619 mg/cigarette

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Total aldehyde 777 mg/cigarette

Side – stream acrolein 100–1700 mg/cigarette

Ambient Urban air acrolein 0002–0.035 mg/m3

Smoky interiors acrolein 0.01–0.05 mg/m3

However, in Table 1, only estimated acrolein concentration values were provided for heated lard

and sunflower oil, these values being 109 and 163 mg/L repectively, and therefore our determined

concentrations of all other alkenals (predominantly trans-2-alkenals such as trans-2-octenal) and the

range of all other aldehyde classifications (including relatively predominant trans,trans-alka-2,4-

dienals, along with lower levels of substituted alkenals and n-alkanals) will undoubtedly provide an

additional and significant contribution to these environmental aldehyde levels, most notably for

consumers with a high incidence of fried food intake. Indeed, the only mention of fried food sources

in Table 1 is codfish fillets, which contained an acrolein content of 100 mg/kg. Notwithstanding,

heated butter was noted to provide low levels of 2-pentenal and 2,4-nonadienal, but this was the only

other peroxidised UFA source of aldehydes mentioned.

Wang et. al. [19] also provided estimates of the mean daily consumption of acrolein form a range

of food sources, and these were cheese (40 µg); donuts (380 µg); codfish fillets (10 µg); wine (1,520

µg); fruits (15 µg); vegetables (250 µg); potatoes (150 µg); and edible cooking oils (10 µg). However,

the latter value will be critically dependent on frying oil sources (those with relatively high levels of

ω-3 fatty acids yielding higher levels of this aldehyde), and also their use and reuse status. From these

8 classes of foods, the average human daily consumption of acrolein alone was estimated to be 2.35

mg. However, since at that time it was known that this LOP was detectable in 35 food classes, the

estimated maximal daily food consumption level in [19] was 5.0 mg/day. Moreover, exposure from

the smoking of tobacco cigarette products (50–100 µg per cigarette) was estimated, which is

equivalent to an additional 1.0–2.0 mg for a human smoking 20 cigarettes per day.

Of the above unsaturated aldehyde estimate, that for the maximal daily human exposure of

acrolein from a combination of food and water sources alone was found to be 0.1 mg/kg BW, along

with an equivalent quantity estimated from tobacco smoking, if appropriate [19].

Consideration of only the estimated non-smoking human contribution, this value in itself is

already 200-fold greater than the ADI value of 0.5 µg/kg BW specified for this aldehyde by the

Australian Government Department of Health (AGDH) [20], which is clearly a major cause for

concern. Dietary sources and estimated dietary intakes of acetaldehyde and formaldehyde, both of

which are also generated in the lipid peroxidation process (from the degradation of secondary

aldehydic LOPs, e.g. MDA for the latter [12]), are provided in Section S3 of the Supplementary

Materials. Moreover, comparative evaluations of the dietary availability for ingestion of aldehydic

LOPS with those of the process toxins acrylamide, monochloropropanediol (MCPD) adduct toxins,

and trans-FAs are discussed in Section S4 (Supplementary Materials).

2.3. Fried food sources of LOPs

Very high concentrations of LOPs, particularly secondary aldehydic ones, are generated during

such processes in view of the autocatalytic, self-propagating nature of this singlet oxygen (1O2)-

catalysed and lipid peroxyl radical (LOO)-mediated peroxidation process [14–17]. Indeed, the total

-unsaturated aldehyde concentration measured in PUFA-rich COs such as sunflower oil

thermally-stressed for a period of 90 min. according to laboratory-simulated shallow frying episodes

(LSSFEs) can reach values as high as 50 mmol/kg [12]. These unsaturated aldehydes are more toxic

than the saturated classes of these compounds which are also generated, and adversely represent 70–

75% of the total aldehyde remaining in COs heated in this manner. Notwithstanding, the very high

total levels of aldehydes often found in used UFA-rich frying oils only represent those retained after

the loss of substantial amounts of them through volatilisation processes, so that they also represent

components of very harmful cooking oil fumes. Health hazards arising from the human inhalation

of such aldehyde-laden fumes in poorly-ventilated kitchen areas are also discussed herein.

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Our research group’s extensive historic investigations of the oxidative deterioration of PUFA-

rich COs during standard frying practices, and their availability for uptake into fried foods such as

potato chips and crisp snacks, etc. available for human ingestion, have been accessible to the scientific

community since 1994 [6]. However, since that time, major advances in the development of

analytical/bioanalytical techniques for the investigation of patterns and concentrations of LOPs in

both food, and biofluid and solid biopsy samples, have been made. Indeed, earlier problems and

complications experienced with the high-resolution NMR analysis of such samples, including

sensitivity issues, have now been largely overcome via the application of newly-developed pulse

sequences, for example. The aldehydic-CHO function regions of the 600 MHz 1H-NMR spectra of a

commercially-available corn oil product exposed to laboratory-simulated shallow frying episodes

(LSSFEs) for periods of 0, 30 and 90 min. are shown in Figure 2, together with a heatmap diagram

displaying the critical dependence of the concentrations of three major aldehydic LOPs generated in

four different COs (of variable SFA, MUFA and PUFA contents) on increasing LSSFE time-points.

These data clearly demonstrate that the heating period-dependent levels of CO aldehydes generated

are high in PUFA-rich oils (corn and sunflower oils), intermediate in MUFA-rich ones (canola oil),

and much lower in SFA-laden coconut oil. More recently, we have confirmed passage of these

secondary LOP toxins from thermally-stressed frying oils into foods fried therein (Figure 3), and have

estimated their contents, which are consistently and considerably greater than those of acrylamide

and monochloro-propanediol (MCPD) adducts [3,14] (Section S4). Indeed, samples of repeatedly-

used frying oils collected from domestic kitchens, fast-food retail outlets and restaurants have

confirmed the generation of aldehydic and further LOP toxins at high concentrations during ‘on-site’

frying practices [3,14]. Our original research studies have been repeated, replicated, and further

exemplified by many research laboratories globally, e.g. [9]. Encouragingly, it now appears that these

highly important public health concerns are appreciated and respected by food science, nutrition and

associated clinical researchers.

(a)

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(b)

Figure 2. (a) Expanded aldehydic-CHO proton (9.20–10.20 ppm) regions of 600 MHz 1H-NMR spectra

of corn oil exposed to laboratory-simulated frying episodes at 180 °C for periods of 0 (blue), 30 (red)

and 90 min. (green). Typical spectra are shown. Abbreviations: -CHO function resonances of 1, trans-

2-alkenals; 2, trans,trans-2,4-alkadienals; 3, 4,5-epoxy-trans-2-alkenals; 4, combined 4-hydroxy and 4-

hydroperoxy-trans-2-alkenals; 5, cis,trans-2,4-alkadienals; 6, n-alkanals; 7, low-molecular-mass short-

chain n-alkanals, particularly propanal and n-butanal from the peroxidation of linolenoylglycerols; 8,

cis-2-alkenals, potentially arising from the thermally-induced isomerism of trans-2-alkenals; 9,

unassigned aldehyde doublet resonance. All resonances visible are doublets, with the exception of

signals 6 and 7, which are triplets (J = 1.73 and 1.74 Hz respectively). Samples were prepared for 1H

NMR analysis by the method described in [11], and spectra were acquired on a JEOL-ECZR600 NMR

spectrometer (De Montfort University facility, Leicester, UK) operating at a frequency of 600.17 MHz.

(b) Heatmap profile showing the time-dependent generation of the three major secondary aldehydic

LOPs, i.e. trans-2-alkenals (t-2-Alken), trans,trans-2,4-alkadienals (t,t-A-2,4-D) and n-alkanals (n-Alk)

in canola (CAO), coconut (COO), extra-virgin olive (OO) and sunflower (SFO) oils exposed to LSSFEs

for periods of 0, 5, 10, 20, 30 60 and 90 min. (ordinate axis codes 00, 05, 10, 20, 30, 60 and 90

respectively). Generalised log- (glog-) transformed aldehyde concentrations (mmol/mol. FA) are

shown on the right-hand side abscissa axis. Deep blue and red colourations depict extremes of low

and high concentrations respectively. The left-hand abscissa axis shows agglomerative hierarchal

clustering of these 3 aldehyde classes, which demonstrate that trans,trans-alka-2,4-dienals, which are

generated only from PUFA peroxidation, have some independence (orthogonality) from a

combination of trans-2-alkenals and n-alkanals, which arise from the fragmentation of both MUFA

and PUFA hydroperoxide sources. Manufacturer-specified SFA, MUFA and PUFA contents of these

oils were 7.5, 63.7 and 28.8% for canola oil; 90.1, 8.1 and 1.8% (w/w) respectively for coconut oil; 13.0,

77.4 and 9.4% for extra virgin olive oil; and 10.3, 29.3 and 60.4% (w/w) for sunflower oil. For canola

oil, 9.8% of the 28.8% (w/w) PUFA content was linolenic acid (as linolenoylglycerols).

Nutrients 2020, 12, 974 9 of 49

(a)

(b)

Figure 3. 1H-NMR Analysis of Aldehydic LOPs in C2HCl3 Extracts of Fast-Food Restaurant Fried Food

Samples. (a) and (b), Expanded aldehydic-CHO proton (9.40–9.90 ppm) regions of the 400 MHz 1H

NMR spectra of C2HCl3 extracts of fried potato chip and chicken (batter portion) servings purchased

from fast-food restaurants, which contain trans-2-alkenal, trans,trans-2,4-alkadienal, 4,5-epoxy-trans-

2-alkenal, combined 4-hydroxy-/4-hydroperoxy-trans-2-alkenal, cis,trans-2,4-alkadienal and n-alkanal

aldehydic LOP resonances in (a), and trans-2-alkenal, trans,trans-2,4-alkadienal and n-alkanal

resonances in (b). Typical spectra are shown. Typically, no aldehydic LOPs were 1H NMR-detectable

in the corresponding meat portion of the fried chicken sample corresponding to the batter extract

spectrum shown in (b). Samples were extracted and prepared for 1H NMR analysis by the method

described in [11], and spectra were acquired on a 400 MHz Bruker Avance NMR spectrometer

equipped with a QNP probe, and operating at 399.93 MHz (De Montfort University facility, Leicester,

UK). Abbreviations: as Figure 1, with F representing formaldehyde in (b).

2.4. Exposure of human populations to fried food sources of dietary aldehydes: rational estimates of their

dietary ingestion from these sources

Unfortunately, global governmental health recommendations for the maximum acceptable

human daily intakes (MHDIs) of aldehydes (i.e. those which are considered to be an acceptable intake

Nutrients 2020, 12, 974 10 of 49

that may be ingested daily throughout an entire lifetime without these agents presenting any

appreciable risk to human health) are either extremely limited or completely unavailable.

However, for acrolein (which is generated from the peroxidation of linolenoylyglycerols, or

alternatively from direct oxidation of glycerol liberated from triacylglycerol backbones via hydrolysis

reactions), the AGDH specified that this value was only 0.5 µg per kg of body weight, i.e. a total of

only 35 g for a mean human body weight of 70 kg [20]. Therefore, the observation that much higher

contents of trans-2-alkenals, trans,trans-2,4-alkadienals and n-alkanals than this limit are present in

fried potato chip servings purchased from a range of fast food ‘take-away/take-out’ restaurants [12]

(Figure 3), indicates a critically-important public health concern.

Using the assumption that all the trans-2-alkenals generated in the frying oils employed by these

outlets is trans-2-octenal (the predominant homologue of this aldehyde classification derived from

the fragmentation of linoleoylglycerol hydroperoxides), its estimated content in what is described as

a ‘large’ 154 g portion of this frequently-consumed fried food is 2.4 mg, a value which is ca. 70-fold

larger than that of this acceptable daily human intake limit for its lower homologue acrolein (which

corresponds to ca. 30-fold greater for its acrolein mass-equivalent figure of 1.04 mg). Parallel estimates

for the most predominant trans,trans-2,4-alkadienal and n-alkanal (n-hexanal) agents produced from

such linoleoylglycerol peroxidation sources were 3.8 and 1.9 mg (acrolein mass-equivalent values of

1.4 and 1.1 mg), respectively, within such a 154 g potato chip portion, and therefore the total aldehyde

content of this typical fast-food source, or at least for that fried in a hypothetical vegetable-derived

frying oil containing 100% (w/w) linoleoylglycerols, is (2.4 + 3.8 + 1.9) mg = 8.1 mg, with acrolein

mass-adjusted values of (1.0 + 1.4 + 1.1) mg = 3.5 mg, of which ca. 70–75% (w/w) are the more toxic

-unsaturated classes. However, it should be noted that the value computed here is estimated from

the consumption of a single staple fried food serving, and also that the above aldehydes are only

three possible, albeit three of the most prevalent, classes of aldehydic LOPs detectable, out of a total

of 10 or more of these generated in UFA-rich culinary vegetable oils during or following standard

frying practices [3,14].

Similarly, assuming that all aldehydes are the most prevalent ones arising from the

fragmentation of oleoylglycerol hydroperoxide (HPM) precursors, estimated potato chip portion

contents of trans-2-alkenal and n-alkanal toxins generated are 2.9 and 2.8 mg respectively (trans,trans-

2,4-alkadienals only arise from the fragmentation of PUFA-derived hydroperoxides) [13]. However,

since oleoylglycerol peroxidation reaction rates are much slower than those of PUFAs, as are the rates

of fragmentation of their HPMs to aldehydes and further products (i.e. it has a substantially lower

peroxidative susceptibility index (PSI) value [1,16]), much lower levels of LOPs are therefore

generated from such sources, and this explains why MUFA-rich oils such as olive oil are relatively

highly resistant to thermally-induced oxidation during standard frying episodes. Indeed,

peroxidative lag-phases for MUFA-rich cooking oils are much longer than those observed with oils

which contain high PUFA contents, e.g. sunflower or corn oils (Figure 2(b)). Several minor (lower

content level) classes of cytotoxic/genotoxic aldehydes are also detectable in fried potato chip samples

(e.g., cis,trans-2,4-alkadienals and formaldehyde), and this also adds significantly to the dietary

aldehydic LOP load.

Importantly, the above estimates pertain to only one 154 g-sized fried potato chip single meal

serving, and those of 300 g, or more are also quite common in the Western diet.

Recently, Grootveld et. al. [3,14] demonstrated that, in addition to the unsaturation status of

frying oils (reflected by their PSI values, which soar with increasing PUFA content), along with a

range of other factors such as frying methods (i.e. deep vs. shallow frying practices), frying

temperatures and durations, for example, the uptake of aldehydic LOP-containing culinary frying

oils (monitored as total lipids through high-resolution 1H-NMR analysis) was a critical determinant

of the aldehyde contents of fried potato chip products. However, the relative molecular content ratios

of trans-2-alkenals, trans,trans-2,4-alkadienals and n-alkanals of these products did not reflect those

present in their frying oil sources (which were also analyzed using 1H-NMR analysis in a C2HCl3

medium), with higher than expected n-alkanal contents. These results are best rationalized in terms

of the higher level of reactivities of the -unsaturated aldehyde classes with potato proteins, amino

Nutrients 2020, 12, 974 11 of 49

acids and further aldehyde-consuming biomolecules, over those of the saturated ones, or more

specifically, the ability of these classes of aldehydes to engage in Michael addition reactions, unlike

their saturated counterparts.

Interestingly, no further updates to the above AGDH ADI value of 0.5 µg per kg of BW, for

acrolein were made in their updated Edition 1/2017 (current as of 31st March 2017). Neither CHPDs,

nor HPMs, were detectable in these C2HCl3 fried food product sample extracts, and this observation

is presumably ascribable to their reactions with potato chip electron donors such as the amino acid

L-cysteine and/or other thiol function-containing biomolecules therein to form less toxic conjugated

hydroxydiene species, and/or their catalytic deterioration to further aldehydes and other secondary

LOP fragmentation products [16]. Indeed, formic acid, a product arising from the degradation of

MDA, was also detectable in these extracts. Therefore, from this investigation, it appears that

commonly-fried food products act as poor sources of CHPDs and HPMs, but nevertheless are rich in

their aldehydic degradation products.

Of further pertinence, shallow-frying episodes generate much higher levels of frying oil LOPs

than deep-frying ones in view of the greater surface area of the oil in the former case, i.e. its much

greater exposure to atmospheric O2 required for the lipid peroxidation process [1,7,14] (Figure 1). For

deep-frying practices, LOPs are only formed on the O2-exposed and -richer surface environment of

the oils employed for this purpose, and subject to a sufficient level of oil admixing/homogenisation

during such processes, are then markedly diluted through their dissipation throughout the much

larger volume bulk oil medium. Interestingly, Totani et. al. [21] found that oxidation was active at the

oil/air interface of bubbles produced by foods being fried in a canola-soybean oil blend according to

deep-frying practices. Marked decreases in the O2 content of these oil blends commenced at a

temperature of 120 °C; however, on being allowed to cool at ambient temperature, a slow restoral of

these pre-diminished O2 levels was found in oil blends pre-heated at a frying temperature of 180oC.

Therefore, it appears that intermittent cooling periods involved in the repeated use of frying oils

during recycling frying episodes facilitate their absorption of atmospheric O2.

2.4.1. Acrolein

Acrolein is generated during the frying, cooking or processing of lipid-containing foods [22–24],

especially those rich in ω-3 FAs such as oily fish products, and also artefactually peroxidised dietary

fish oil provisions or supplements in general [25]. An estimated mean concentration of acrolein of

0.51 mmol/kg was found in samples of five types of cooking oil heated to 80oC and aerated for a

period of 20 hr. [26]. Notably, in view of its high volatility, this unsaturated aldehyde was detected

in emissions arising from n = 4 heated cooking oil products in China [27] at levels varying from 49

µg/L in peanut oil to 392 µg/L in rapeseed oil (the latter oil has a relatively high content of the ω-3

FA linolenic acid (as linolenoylglycerols), one major PUFA source of this aldehyde). It should also be

noted that selected ingredients present in commercially-available breading systems and batter can

also give rise to acrolein in fried food matrices [24].

2.4.2. HNE and HHE

Estimates of the concentrations of HNE alone in French fry samples collected from n = 6 U.S.

fast-food restaurants [28] were found to range from 8 to 32 g/100 g (0.51 to 2.05 mol/kg), values

corresponding to 12–50 g for a standard ‘large’ sized 154 g serving. Moreover, assuming a mean

frying oil uptake of 12% (w/w) (range 1-33% (w/w) [3]), our laboratory’s 1H-NMR-based estimate of

the mean HNE content of 154 g potato chip portions is ca. 30 µg, a value which is in very good

agreement with those found in [28] (assuming no chemical reactions of this LOP with potato chip

biomolecules, e.g. proteins and amino acids, which is, however, unlikely). Furthermore, these

determined values are not dissimilar to the above Korean estimates. Our estimates have also

confirmed that HNE accounts for only ≤ 1% of the total molar -unsaturated aldehyde content of

fried potato chips (relative amounts of 4-hydroxy-trans-2-alkenals found in thermally-stressed

sunflower, corn and canola frying oils were <10% of the total measured [12]). Moreover, HNE levels

previously determined in sunflower oil were found to be ca. 350 and 430 mol./L when it was

Nutrients 2020, 12, 974 12 of 49

thermally-stressed at 190 °C for prolonged 17.5 and 20.0 hr. durations, respectively [29]. These large

differences observed between the HNE contents of fried potato chips and the oils in which they are

fried are presumably explicable by the higher reactivity of 4-hydroxy-trans-2-alkenals with HNE-

scavenging potato chip biomolecules than that of trans-2-alkenals and n-alkanals, and/or an enhanced

level of their degradation or further oxidation therein when expressed relative to those for the other

aldehyde classes detectable. Human exposure to 4-hydroxy-trans-2-alkenals in vegetable frying oils, fish and shellfish in

Korean diets has been previously assessed using GC/MS/SIM as an analytical strategy, along with

National Health and Nutrition Survey data to evaluate dietary intake patterns [30]. From these

results, the combined HNE and HHE exposure was estimated to be only 16.1 µg per day

(approximately 75% of which was HNE), i.e. 0.3 g/kg for a mean Korean human body weight of 60

kg. Notwithstanding, the risks posed to humans could not be determined, despite the known

toxicological actions of these aldehydes. However, on consideration of their basal tissue

concentrations, the researchers involved concluded that the dietary availability of such agents may

not present a significant human health risk.

2.5. Risk assessments of aldehyde intake in humans: Estimated margin of exposure (MOE) values

Acceptable daily intake (ADI) is a very important parameter for the evaluation of risks to

humans presented by dietary and environmental toxins, for example, and represents the maximum

amount of a chemical substance that can be ingested on a daily basis throughout an entire lifetime

with no appreciable health risk. For food additives or contaminants, this ADI parameter is usually

computed and then employed to determine its risk status via comparisons of it to mean and

associated confidence interval values for estimated human exposure and/or intake levels. However,

for food contaminants and additives, the ADI may also be termed the tolerable daily intake value

(TDI) value.

ADI values are usually obtained from the lowest no observed effect level (NOAEL), which is

derived from long-term in vivo animal model investigations. Hence, such ADI indices arise from the

application of a safety or uncertainty factor to the NOAEL value of the most sensitive testing species.

This safety factor, which is most commonly 100, is applied in view of the requirement to allow for

‘between-species’ differences and variabilities, and also those featured in their toxicokinetic and

toxicodynamic properties. As an example, and for the purpose of comparative evaluations with

dietary aldehydes, in 2010 Tardiff et. al. [31] performed a safety evaluation of ingested acrylamide

using a ‘state-of-the-art’ physiologically-based toxicokinetic model, and TDI (ADI) values for this

food toxin was found to be 40 µg/kg BW per day (equivalent to 2.8 mg for a 70 kg BW human), but

for cancer only 2.6 and 16 µg/kg BW per day for this agent and its glycidamide metabolite respectively

(equivalent to only 182 µg and 1.12 mg/day respectively for a mean 70 kg BW human). The margin

of exposure (MOE) values (equation 1) of aldehydes, and LOPs in general, should be employed in

risk determinations, since these consider the benchmark dose lower confidence limit (BMDL10), a

parameter which represents the lower 95% confidence interval limit of the amount (dose) of an

aldehyde to give rise to the occurrence of a toxic effect when expressed relative to that of a control:

MOE = BMDL10 (µg/kg BW/day)/EDI (µg/kg BW/day) (1)

For acrolein, acetaldehyde and formaldehyde, these BMDL10 values are 360 [32], 5,600 [33] and

2,800 µg/kg BW/day [34] respectively. These values have been previously documented by Ferreira et.

al. [35], and Peterle et. al. [36].

On the basis of these figures, estimated MOE values are 360/33.6 = 10.7 for acrolein; 5,600/137 =

40.9 (Europe) and 5,600/274 = 20.4 (USA) for acetaldehyde; and 2,800/(21–200) = 14 to 133 for

formaldehyde. EDI values for acrolein, acetaldehyde and formaldehyde were obtained from Wang

et. al. [19], [37] and [38] respectively. MOE values which are lower than a value of 10,000 indicate a

potential WHO-defined health risk [39].

For the purpose of this Commentary paper, we have also estimated MOE values for the potato

chip contents of the most predominant aldehydes derived from the thermo-oxidation of

Nutrients 2020, 12, 974 13 of 49

linoleoylglycerols, and the fragmentation of their hydroperoxides (Table 2). These estimates were

computed on the assumption of humans consuming a single specified potato chip servings daily, but

these can be readily adjusted to those consuming averages of 2 or 4 such servings per week my

multiplying by the 2/7 and 4/7 factors for amounts available, and corresponding 7/2 and 7/4 ones for

MOE estimates, respectively. Clearly, these estimates are lower or strikingly lower than the WHO

limit of 10,000 reported (especially for the α,β-unsaturated aldehydes), even when considering an

average two portion intake per week.

Table 2. Toxicological MOE Indices for Linoleoylglycerol Hydroperoxide-Derived Aldehydes in

Fried Potato Chips.

Aldehyde Classification

Potato Chip Serving

Size (g): trans-2-Octenal

trans,trans-Deca-2,4-

dienal n-Hexanal

71 g 1.09 (0.48)

MOE: 52.5

1.73 (0.64)

MOE: 39.4

0.88 (0.50)

MOE: 784

154 g 2.37 (1.04)

MOE: 22.7

3.76 (1.40)

MOE: 16.9

1.91 (1.08)

MOE: 363

300 g 4.61 (2.01)

MOE: 12.5

7.32 (2.72)

MOE: 9.2

3.73 (2.10)

MOE; 187

Estimated Aldehyde

Content (ppm): 15.3 (6.8) 24.4 (9.0) 12.5 (7.0)

Estimated amounts of aldehydes (mg) and contents (ppm) for typical fried potato chip portion

sizes of 71, 154 and 400 g (proportionate acrolein mass-equivalent values are provided in brackets).

These values correspond to the most predominant aldehydes derived from the thermo-oxidation of

linoleoylglycerols. Margin of exposure (MOE) values for aldehyde contents were estimated using the

acrolein-equivalent mass values only, and assuming that each potato chip portion represented a

mean daily intake for those with a high level of fried food intake. The BMDL10 value of acrolein was

used for the trans-2-octenal and trans,trans-deca-2,4-dienal estimates, and that for acetaldehyde was

used for the n-hexanal one.

3. Fate and Adverse Health Effects of Primary and Secondary LOPs in Humans and Animal Model

Systems Following Dietary Ingestion

3.1. Lipid hydroperoxide aldehyde precursors (CHPDs and HPMs): Gastrointestinal interactions, metabolism

and biotransformations, in vivo absorption, toxicity and deleterious health effects

Lipid hydroperoxides can potentially give rise to a series of intestinal disorders, including

colorectal cancer [40], and their ability to interfere with both molecular and cellular processes, and

hence exert a clinically significant impact on intestinal integrity, is responsible for these actions.

Historically, investigations focused on an exploration of the acute toxicity of highly purified

methyl linoleic hydroperoxide (MLH) were performed by Cortesi and Privett as early as 1972 [41].

Indeed, the median lethal dose (MLD) value of intravenously (i.v.)-injected MLH was found to be

0.70 mmol/kg (ca. 230 mg/kg) of body weight (BW) in adult male rats. However, single oral dosages

of this agent, which were 10-fold higher than those administered via the i.v. route, gave rise to no

observable deaths in these experimental animals, an observation indicating either their failure to be

absorbed in vivo, or their metabolic modification within the gastrointestinal (GI) tract (e.g., reduction

to conjugated hydroxydiene species of a much lowered toxicity), accounted for this phenomenon. For

the i.v.-treated animals, the major adverse effect observed was localized within the lungs, which

enlarged from fluid accumulation and oedema; fatalities therefore arose from severe lung congestion

and injury.

However, it is important to note that since the above level of orally-administered MLH far

exceeds that of the estimated daily human intake of lipid hydroperoxides, which is 1.5 mmol/kg

(equivalent to 21.4 µmol/kg BW) [42].

Nutrients 2020, 12, 974 14 of 49

In an earlier study [43], daily i.v. injections of a more realistic, lower dose of MLH (50 mg/day),

or its continuous infusion at a rate of 206 g/min., to experimental rabbits, was found to markedly

diminish their -TOH stores. Following 10–14 days, the injected animals displayed significant fatty

degeneration and necrosis of the liver, along with creatinuria, and an accelerated muscular

incoordination over those of an untreated control group. Although the creatinuria was circumvented

by the oral administration of very high doses of -TOH (100 mg/day), glutathione peroxidase-

replenishing selenite exerted no blocking influence on both creatinuria and liver lesion incidence. For

the MLH-infused group of animals, an elevated erythrocyte fragility and substantial creatinuria were

observed. Therefore, chronic administration of these low MLH doses gave rise to a rapid

consumption/degeneration of endogenous -TOH, together with an increased incidence of deficiency

symptoms for this antioxidant.

A further study explored the metabolic transformations of orally-administered lipid

hydroperoxides in carp both in vitro and in vivo [44]. Analysis of methyl ester reaction product

derivatives arising from equilibration of 13-hydroperoxy-cis-9,trans-11-octadecadienoic acid with

carp ‘acetone powder’ in vitro revealed that methyl 13-oxo-cis-9,trans-11-octadecadienate, methyl 13-

hydroxy-cis-9,trans-11-octadeca-dienoate, methyl 11-hydroxy-trans-12,13-epoxy-9-cis-octadecenoate,

and methyl 9-hydroxy-trans-12,13-epoxy-trans-10-octadecenoate were the four major metabolites

identified, i.e. one oxodiene and one hydroxydiene species from redox routes, and two hydroxy-

epoxy acids. Oral administration of U-14C-labeled MLH to carp at a level of 0.10 mL/100 g, equivalent

to a very high dose of 2.68 mmol./kg, demonstrated that the predominant metabolites found in

selected organs were hydroxy-octadecadienoate and oxo-octadecadienoate, with ca. 8% of the dosed 14C radiolabel remaining in the body following a 24 hr. period. Since hydroperoxy-octadecadienoates

were found to be absent from carp organ lipid profiles, these data indicate that linoleate-derived

CHPDs (the most common dietary CHPDs) are firstly intestinally redox-transformed to their

corresponding hydroxy- and oxo-adducts, and are then absorbed into the fish circulatory system

where they have access to essential organs and tissues. Hence, these observations suggest that

although CHPDs are not absorbed in vivo, their intestinal primary redox metabolism products are, at

least in carp. These observations are supported by further experimental animal system investigations

described below.

Additionally, Kanazawa and Ashida [45] studied the catabolic fate of linoleic acid

hydroperoxide in a rat GI system in order to determine the molecular nature of LOPs derived from

this source, and which are absorbed into the systemic circulation. Low, albeit perhaps dietarily-

relevant concentrations of this primary LOP (6.5 or 18 mol/L) failed to penetrate into the intestines

(presumably because of its rapid biochemical consumption prior to reaching this site), whereas

higher doses (200 or 800 mol/L) partially leached into this environment. In 14C radiolabel

investigations, products generated therefrom comprised conjugated hydroxydienes (~ 4%), epoxy-

ketones (~ 10%), aldehydes (~ 2.4%), and ~ 13% unidentified 14C-labelled species, along with 27% of

the unmodified peroxide substrate. However, gastric tissue took up 25% of the label, and ca. 6% was

found in the intestinal lumen and tissue as degraded aldehydes. Administration of an aldehyde

mixture dose gave rise to the accumulation of significant amounts of HNE (Section S5), and the less

toxic saturated aldehyde n-hexanal in the liver after a 15 hr. duration (both these aldehydes are

known to specifically arise from the fragmentation of linoleic acid hydroperoxides). Therefore,

evidence for the degradation of linoleic acid hydroperoxide to aldehydes in the stomach was

provided by this study, and the researchers involved concluded that such secondary aldehydic LOPs

are partially absorbed into the circulation.

Fortunately, the human intestinal system is set up with a battery of defense mechanisms to

counter the toxicological onslaught of CHPDs from both endogenous and dietary sources, along with

other ROS. Such defense barriers include peroxide-scavenging catalase, superoxide dismutase (SOD)

and, most importantly for lipid hydroperoxides, the hydroperoxide-neutralising electron-donor thiol

compound glutathione (GSH) and its peroxidase enzymes (GPx) [46–48]. Intriguingly, the GI tissue

network is the only one which has the ability to express all four classes of GPx enzymes

simultaneously, and the sole expression of GI-GPx in this system has indicated that it may be

Nutrients 2020, 12, 974 15 of 49

exclusively targeted to protect against the adverse in vivo absorption of dietary lipid hydroperoxides,

and peroxides in general [49].

Interestingly, GI GPx blocks the shuttling of lipid hydroperoxides in CaCo-2 cells [50], which are

of much value to intestinal absorption investigations since they differentiate to form a polarized

epithelial cell monolayer serving as a physical and biochemical hurdle to low-molecular-mass

molecules and ions. Kanner and Lapidot [51] were the first to demonstrate that ingested PUFAs were

peroxidised within the gut, and for this purpose they investigated free radical-mediated processes

taking place in the stomach’s acidotic environment which could, in principle, promote the generation

of CHPDs from these precursors, along with the concomitant oxidation of further dietary substrates.

Their results suggested that human gastric fluid serves as a highly appropriate ‘bioreactor’ matrix for

accelerating the peroxidation of dietary PUFAs and additional dietary constituents, and also the

potential harmful actions of ingested CHPD LOPs. Moreover, they also found that such localized

stomach-based oxidation was completely suppressed by the inclusion of plant-derived dietary chain-

breaking antioxidants, an observation which demonstrates the protective actions of such agents, and

their beneficial health effects in vivo.

Similarly, Tullberg et. al. [52] explored the oxidation of cod liver oil lipids during GI digestion,

using models involving standardised digestion protocol-matched human digestive juice, and porcine

bile and digestive juice media; fish oil mixed with water at a level of 0.13 mg/mL was employed as

an initial meal. Malondialdehyde (MDA), HNE and 4-hydroxy-trans-2-hexenal (HHE) were analysed

in these digests (using liquid chromatography/atmospheric pressure chemical ionization-mass

spectrometry), as were free fatty acids (FAs) by gas chromatography-mass spectrometry (GC-MS); HHE

specifically arises from the peroxidation of -3 FA sources. Results acquired showed that although

aldehydic LOP generation was low during gastric digestion, it was enhanced in the duodenal digestive

process. Aldehyde generation was accelerated when using human digestive juices over that found using

the porcine system. Free FA liberation was only detectable during the intestinal phase of the protocol, and

this parameter attained values of up to ca. 30%.

Interestingly, stable hydroxymonoenes and conjugated hydroxydienes generated from the GI-

based reduction of lipid hydroperoxides are also available in the human diet, and, like aldehydes,

can also be absorbed from the gut into the systemic circulation [53]. Therefore, there is no shortage of

controversy regarding their measurement in biofluids and tissues as biomarkers of ‘oxidative stress’

in vivo.

However, despite these considerations, it appears that fried foods, which represent one major

source of dietary LOPs, contain little or no lipid hydroperoxide precursors of aldehydes [12], which

as noted above also exert a range of toxicological effects when administered via the i.v. route in animal

model studies [41], and also in in vitro evaluations. Such secondary aldehydic LOPs are more stable

than CHPDs and HPMs when introduced into complex food and biological matrices (the latter

including human biofluids and tissues), which both contain relatively high levels of many LOP-

reactive scavenging agents, including hydroperoxide function-reducing electron donors, and

aldehyde-consuming amine and thiol functions present in a wide variety of biomolecules of both

low- and high-molecular-mass. Moreover, in vivo, enzymes available for the redox interconversion of

hydroperoxides to hydroxydienes, along with the oxidation and/or reduction of aldehydes to their

corresponding carboxylic acid and alcohol adducts, respectively, are readily available. Additionally,

such aldehydes may reversibly react with food alcohols and/or carbohydrates to from hemiacetals

and acetals.

3.2. Secondary aldehydic LOPs: Dietary ingestion, gastrointestinal fate, in vivo absorption, metabolism and

toxicological effects

The molecular nature, toxicities and health hazards potentially presented by aldehydic LOP

toxins have been previously explored in some detail, as have analytical strategies available for their

determination and monitoring, e.g. in fried food sources, and human/animal biofluids and tissues,

for probing their in vivo absorption, biodistribution, metabolism and urinary excretion (an example

of the 1H NMR analysis of aldehydes, specifically LOPs and vanillin, in a typical non-fried food

Nutrients 2020, 12, 974 16 of 49

product is shown in Figure 4). Indeed, the toxicological and pathogenic properties conceivably

arising from the ingestion of aldehydic LOP-containing COs heated according to standard frying

practices (in the form of CO-absorbing fried foods for humans), and also aldehyde model systems,

include their potential roles in the development and perpetuation of cardiovascular diseases [54–56],

their carcinogenic [57–61], gastropathic [62], pro-inflammatory [63], and teratogenic properties [64],

contributions towards neurodegenerative disorders [65], their hypertensive effects [66]; the

development and perpetuation of diabetes [67];and respiratory and pulmonary complications, the

latter especially for acrolein [68]; this list is inexhaustive. The inhalation of volatile aldehydes and

other carbonyl compounds by workers employed in poorly-ventilated fast-food/restaurant retail

outlets is also considered to pose a major threat to human health [69], particularly with reference to

established links between an increased incidence of lung cancer and cooking oil fume inhalation

amongst such personnel [13,70–72]. Indeed, since a wide range of aldehydic LOPs such as acrolein

(the lowest homologue trans-2-alkenal) have boiling-points (b.pts) below or far below standard frying

temperatures (ca. 180 °C), cooking oil fumes are very rich indoor air pollutant sources of these toxins.

(a)

Nutrients 2020, 12, 974 17 of 49

(b)

(c)

Nutrients 2020, 12, 974 18 of 49

(d)

(e)

Figure 4. 600 MHz 1D 1H and 2D 1H-1H correlation spectroscopy (COSY) NMR spectral profiles of a

C2HCl3 extract of a commercially-available chocolate hazelnut spread product. (a) Expanded 9.40–

9.90 ppm region of a 1D spectrum of this extract showing an intense –CHO function resonance arising

from the flavouring agent vanillin (abbreviated V1), along with 1H-NMR-detectable traces of

trans,trans-2,4-alkadienals (2) and long-chain n-alkanals (6). (b) Expanded 5.655–6.670 (F1 axis) and

9.364–9.660 ppm (F2 axis) region of a 1H-1H COSY spectrum acquired on this extract, revealing

connectivities between the C1-CHO and C2-CH=CH- resonances of trans,trans-2,4-alkadienals. (c)

Expanded 2.230–2.670 (F1 axis) and 9.668–9.797 ppm (F2 axis) region of the 1H-1H COSY spectrum

shown in (b), showing differential molecular couplings between one major (A) and one relatively

minor (A1) long-chain n-alkanal species. (d) and (e), Expanded 5.7–8.2 and 3.4–4.2 ppm regions of the

1D spectrum shown in (a) respectively, with resonances ascribable to the C5H/C6H (V2) and C2H

(V3) aromatic, and C3-OCH3 (V4) protons of vanillin indicated. DV represents a tentative assignment

to the C3-OCH3 function of divanillin, a vanillin oxidation product. Further abbreviations: -OOH,

lipid hydroperoxide-OOH function resonance; CHCl3, residual chloroform; X, residual chloroform 13C satellite.

Nutrients 2020, 12, 974 19 of 49

3.2.1. In vivo absorption of and metabolic/biotransformation routes for aldehydic LOPs

The GI tract is continually exposed to toxic aldehydes, and subsequent to digestion they are

absorbed into the lymphatic system, or directly into the systemic circulation [73]. Indeed, in 1998, our

laboratory demonstrated that typical trans-2-alkenals generated during the thermal stressing of

PUFA-containing frying oils (trans-2-pentenal and -nonenal) are indeed absorbed from the gut into

the systemic circulation in vivo, then metabolised by a process involving the primary addition of GSH

across their electrophilic carbon-carbon double bonds, and finally excreted in the urine as C-3

mercapturate alcohol derivatives, i.e. as N-acetyl-S-(3-hydroxypentyl)-L-cysteine and -(3-hydroxy-

nonyl)-L-cysteine derivatives, respectively, in experimental rats [73]. However, the administered

levels of these aldehydes were as high as 10 and 100 mg/kg. Generation of these metabolites also

involves reduction of their chemically-reactive aldehyde/aldehyde hydrate (-CHO/-CH(OH)2)

functions to primary alcohol species via the actions of hepatic alcohol dehydrogenase. These results

were consistent with the findings made in [50], which provided evidence for the at least partial

absorption of such aldehydes into the circulation.

However, it should also be noted that this study found that at a 16 hr. post-dosing time-point,

approximately 15% of the orally-administered dose of trans-2-nonenal was oxidatively transformed

to its corresponding carboxylic acid metabolite within the stomach [73].

Consistently, following the subcutaneous injection of the simplest trans-2-alkenal acrolein to

rats, N-acetyl-S-(3-hydroxypropyl)-L-cysteine was detected and isolated as a key urinary excretion

product [74], and these results ae also fully consistent with our 1H NMR-based urinary metabolic

screening investigations [73], including the hepatic metabolic reduction of the aldehyde functions to

alcohol derivatives. However, for these experiments, acrolein was administered by the subcutaneous

injection of a 1% (v/v) solution in arachis (peanut) oil into the lumbar region; the vehicle may itself

have served as a source of aldehydic LOPs, especially if allowed to peroxidise during periods of

storage or solution preparation.

In a scientifically elegant and highly informative early study published in 1985, McGirr et. al.

[75] found that a significantly high proportion of dietary MDA is covalently linked to dietary

proteins, and an acid-labile urinary metabolite (the N-acetyl derivative of the lysine-MDA enaminal

N-(2-propenal) lysine) was detectable in experimental rats following oral administration of a serum

albumin-MDA adduct at a level of 2 mg MDA equivalents/kg BW. Furthermore, this compound was

also demonstrated to be a major urinary metabolite of this dialdehyde administered as its sodium

enolate salt via stomach intubation. Elevated concentrations of this metabolite were excreted by rats

fed a diet rich in highly-peroxidisable cod liver oil. However, these researchers were also able to

identify low levels of this metabolite in the urine of fasted rats, and this observation provided

evidence that it is also formed as a product derived from the in vivo peroxidation of PUFAs, in

addition to its ingestion as a dietary LOP (such as those formed during high temperature frying

practices in the human diet), or alternatively, through the prolonged storage of PUFA-containing

foods. Injection of MDA as its sodium enolate salt to fasted animals markedly increased its urinary

concentration, as expected. In view of the acid lability of N-(2-propenal) lysine, it is possible that free

MDA may be liberated from this primary Schiff base product, and perhaps also from more prevalent

dietary protein lysyl residue adducts, in the GI tract (particularly the stomach), so that it may be

ingested into the systemic circulation as a free (non-adducted) agent.

One recent key investigation appears to have resolved the longstanding critical question

regarding whether there is some clinically-significant in vivo absorption of 4-hydroxy-trans-2-

alkenals, potentially one of the most toxic classes of -unsaturated aldehydes available in human

dietary sources [76]. Details of this study are provided in Section S5 (Supplementary Materials).

Since HNE is universally considered to represent a very important secondary LOP, its metabolic

fate has been extensively investigated. An exhaustive review of the roles of 4-HNE in health and

disease is provided in [77], including a detailed evaluation of its metabolic and biotransformation

products. However, important examples of studies of its metabolic fate both in vivo and in vitro are

also provided in section S5 of the Supplementary Materials. Interestingly. HNE-modified proteins

Nutrients 2020, 12, 974 20 of 49

also appear to be key features of metabolic diseases, and hence offer potential to serve as effective

biomarkers for such conditions [78].

3.2.2. Associations between dietary fried food aldehyde concentration patterns and those of human

blood plasma: Potential tracking of dietary LOPS in vivo?

In 2000, Mak et. al. [79] determined a total of 22 individual aldehydes in circulating arterial blood

plasma samples collected from n = 8 patients with congestive heart failure (CHF), along with those

from an equivalent number of age-matched participants with normal left ventricle (LV) function, i.e.

non-CHF controls. Aldehydes were determined via a GC/MS bioanalytical strategy, and these

included long- and short-chain n-alkanals, trans-2-alkenals, 4-hydroxy-trans-2-alkenals, trans,trans-

2,4-alkadienals, MDA and the dietary flavouring agent furfural. Mean plasma concentrations, or

ranges for the mean aldehyde concentration values of specific structural homologues within each

class, are provided in Table 3 for both control and CHF groups, as are full ranges for the individual

sampling values found in n = 36 samples of potato chips collected from fast-food/take-away

restaurant outlets.

Table 3. Mean concentrations, or concentration ranges of these mean values (nmol/L) of aldehydes

determined in the blood plasma of n = 8 patients with congestive heart failure (CHF) and n = 8 age-

matched normal LV function controls by a GC-MS technique (adapted from [79]).

Non-CHF Controls

(nmol/L)

CHF Disease

(nmol/L)

Fried Potato Chips

(μmol/kg)

Long-Chain n-Alkanals (7) 69–573 42–339 19–560

Short-Chain n-Alkanals (1) 67 91 nd

trans-2-Alkenals (4) 106–527 163–874 0–430

4-Hydroxy-trans-2-Alkenals

(4) 33–211 16–434 0.5–2.1 [68]

trans,trans-2,4-Alkadienals

(2) 152–180 148–420 0–443

Malondialdehyde (MDA) 96 101 0–6 *

Furfural 2,450 4,060 nd

The bracketed numbers in the first (molecular classification) column refer to the number of aldehydes

included for each classification specified for the blood plasma samples analysed. Also listed are the

ranges of contents (µmol/kg) found for samples of fried potato chips (or French fries) purchased from

fast-food/take-out restaurants (long- and short-chain n-alkanals, trans-2-alkenals, and trans,trans-alka-

2,4-dienals were determined by our 1H-NMR analysis approach, but those for 4-hydroxy-trans-2-

alkenals are those reported in Ref. [68] using an LC-MS method. However, both 4-hydroxy-trans-2-

alkenals and furfural are also readily 1H-NMR-detectable and quantifiable. * MDA was specifically

determined by a modification of the method outlined in [6], which involved the reaction of

thiobarbituric acid (TBA) with this dialdehyde to form a pink/red chromophoric derivative, but only

subsequent to its relatively specific extraction into an aqueous medium (mean ± SD first extraction

efficacy: 78 ± 2%).

The blood plasma results acquired in [79] demonstrated that CHF patients had significantly

higher levels of total aldehydes, together with a range of unsaturated ones (specifically, trans-2-

alkenals and 4-hydroxy-trans-2-alkenals, the latter including HHE and HNE), and furfural.

Conversely, the normal LV function control group involved had significantly higher levels of n-

alkanals over those of the CHF patients. Furthermore, the dietary flavouring agent furfural was by

far the most predominant aldehyde present, i.e. 37 and 44% of the total aldehydes determined in

control subjects and CHF participants respectively) and was found be significantly upregulated in

the latter. Furfural is not a LOP, but in addition to its potential genotoxic and carcinogenic properties

[73], this food flavourant has been shown to give rise to the accumulation of ROS and cellular damage

in Saccharomyces cerevisiae [80].

Nutrients 2020, 12, 974 21 of 49

However, aldehydes of the 2,4-alkadienal class monitored in these samples only featured

trans,trans-hepta- and trans,trans-2,4-nonadienals, and the only other di-unsaturated aldehyde

monitored was trans,trans-2,6-nonadienal. Moreover, cis- and trans-deca-4-enals were measured as a

combined sum. Additionally, this study was complicated by (1) the very high incidences of

comorbidities in the male participants recruited to it (mean within-group ages ca. 60 years),

specifically diabetes, hypertension, and hyperchloesterolemia in both groupings, and (2) medical

therapies received by them, i.e. β-blockers, nitrates, ACE inhibitors and calcium channel blockers in

both groups, and additionally diuretics in the CHF one. Notably, all vitamin supplements were

withheld from participants for a minimum duration of 7 days prior the study, and all oral medications

were withheld on the morning of the investigation.

From these results, we therefore elected to perform a comparative statistical evaluation of these

blood plasma LOP profiles in terms of the mean molar levels of different classes of aldehydes

determined therein expressed as a proportion of the total LOP-relevant aldehyde concentration found

in the samples analysed, i.e. those within the above control and CHF groups, to those of the same

mean molar ratios of the aldehyde classification contents found in frequently-consumed fried potato

chip samples collected from fast-food restaurants (Table 2 and Table 3), specifically those fried in

commonly-utilized vegetable oil frying media, as noted in Section 2.3 above. The use of molecular

ratio variables for this exercise is, however, quite fortuitous, since they are expected to be less

sensitive to the potential influences of a range of latent generic variables such as participant BMIs,

ages, etc.

For this purpose, blood plasma levels of furfural were excluded from the computation of

proportionate aldehyde contents since it is not a LOP, and nor was it detectable in any of the fried

potato chip samples analysed by 1H-NMR analysis Unfortunately, it was also not possible to compute

the relative proportions of alka-2,4-dienals in the above two blood plasma groups, since trans,trans-

2,4-decadienal, the major trans,trans-2,4-alkadienal arising from the peroxidative deterioration of

linoleoylglycerols (Section S2), was not determined in [79], and neither was HHE, the major 4-

hydroxy-trans-2-alkenal derived from the decomposition of CHPDs generated from the oxidation of

-3 FAs, e.g. -linolenoylglycerols. Short-chain aldehyde concentrations provided in this report were

those for n-butanal only – since this was the only such analyte included, these values were also

removed from the dataset prior to statistical analysis, although they do remain valuable, since such

aldehydes predominantly arise from the peroxidation of -3 FAs [12]. Therefore, each proportionate

aldehyde class considered comprised those of n-alkanals, trans-2-alkenals, 4-hydroxy-trans-2-

alkenals and MDA only, and all proportions computed represented the concentrations of each of

these LOPs divided by the sum total of them, plus those of all possible alkadienals found. In view of

these limitations, results obtained from these comparative evaluations should be treated with some

caution.

The mean relative proportions (ratios) of the concentrations of long-chain n-alkanals:trans-2-

alkenals:4-hydroxy-trans-2-alkenals:MDA in these three groups of samples were compared and

statistically tested for any significant differences between them. Expressed as percentages of the total

aldehydes detectable (minus contributions from furfural), these ratios were: 40:31:0.20:0.60 for fried

potato chips (mean percentages for a newly-acquired 1H-NMR dataset, n = 36); 46:30:9:2 for normal

LV function (control) subject blood plasma; and 26:39:17:2 for CHF patient blood plasma. Direct

comparison of these proportions for the potato chip sample profiles with those of the control blood

plasma group showed that although the trans-2-alkenal and, to a lesser extent, long-chain n-alkanal

values were quite similar for this comparison, those of 4-hydroxy-trans-2-alkenals were much

elevated in the latter, and these data indicate that, in addition to post-ingestional, aldehyde class-

dependent modifying factors such as differential rates and extents of their absorption, metabolism,

chemical reactivity, protein adduct formation and biodistribution, etc. between each aldehyde class

considered, this aldehyde classification appears to arise from in vivo peroxidation processes.

Moreover, although the proportionate MDA levels remained small for both these groups, such mean

values were elevated approximately 4-fold in the normal LV function blood plasma one.

Nutrients 2020, 12, 974 22 of 49

However, a further major consideration is the dietary availability of all aldehydes considered,

i.e. what proportion of them are ‘free’ and what are constituted as adducts with food proteins (as

noted for MDA [75]), alternative biomacromolecules, or low-molecular-mass nutrient metabolites

such as free amino acids?; such adducts may represent latent sources of these toxins, which may be

liberated within the GI system, for example. Notably, our laboratory determines the ‘free’, non-

adducted form of these aldehydes in fried food products, and hence our estimated values (Table 2

and Table 3) will presumably represent underestimates of the total taken up from COs during frying

practices.

A permutation testing strategy performed via partial redundancy analysis (PRDA) on the log10-

transformed proportionate aldehyde level dataset (involving 104 permutations) revealed that

aldehyde classification-conditioned differences observed between the three sample groups were

statistically significant (p = 0.049), as indeed were those ‘between-aldehyde classifications’ (p = 0.009),

the latter being expected, of course (the log10-transformation was required to counteract within-

sample negative correlations between proportionate/percentage variables). These significant

differences were clearly manifested by 4-hydroxy-trans-2-alkenals and MDA being much greater in

the normal LV function (control) blood plasma profiles over those of fried potato chips. However,

they also arise from the CHF blood plasma group having upregulated proportionate trans-2-alkenal

and 4-hydroxy-trans-2-alkenal levels (over both the control plasma and potato chip serving groups),

and significantly higher proportionate MDA concentrations than the fried potato chip group. This

significant ‘’between-sample group’ effect observed is also explicable by the large differences

observed between the proportionate levels of total n-alkanals between the CHF group and the two

others compared.

Therefore, the observation of very similar fractional aldehyde contents of both n-alkanals and

trans-2-alkenals in the large potato chip and smaller control blood plasma sampling groups may serve

to indicate that such LOPs are dietary-derived. If this is the case, then the in vivo ‘conservation’ of

their proportionate levels may also reflect the overall lower, albeit differential chemical/biochemical

reactivities of these classes of aldehydes than those of 4-hydroxy-trans-2-alkenals and MDA,

following their ingestion by humans. Of particular note, in vivo, n-alkanals serve as substrates for

pyruvate dehydrogenase, but -unsaturated aldehydes are not affected by this enzyme [81].

Moreover, as noted above, unsaturated aldehydes readily take part in Michael addition reactions

with GSH to form their primary detoxification GSH conjugate products [82,83], but n-alkanals clearly

do not (although they may form Schiff base adducts with the terminal amino function of this

tripeptide). However, the rather substantial differences observed between these two groups’

proportionate 4-hydroxy-trans-2-alkenal and MDA concentrations certainly indicate, but do not

confirm, that such toxins may be generated from in vivo lipid peroxidation processes.

Proportionate total 4-hydroxy-trans-2-alkenal levels in the CHF blood plasma group were also

significantly greater than those of the normal LV function control group (ca. 2-fold), but this

observation was reversed for long-chain n-alkanal concentrations, the latter results being consistent

with our proportionate levels estimated in fried potato chips. The markedly elevated proportionate

value of CHF blood plasma levels of the former class of aldehydes may have been expected in view

of an enhanced level of in vivo oxidative stress associated with this condition. However, if this was

the case, why was it that the mean level of furfural, a non-LOP dietary flavouring agent, was also

significantly increased from 2.45 µmol/L in the control group to 4.06 µmol/L in the CHF one (p <

0.01)? Possibly these differences are also partially explicable by differing dietary regimens between

these two groups, perhaps an increased level of aldehyde-loaded fried food consumption and/or an

enhanced furfural intake in the latter (this flavouring agent is readily absorbed subsequent to

administration by any route [84])? Study participants were not fasted for a minimum required period

prior to the collection of blood samples in this study, so it certainly appears that such aldehydes may

at least partially arise from such dietary, or perhaps alternative exogenous sources. Further possible

limitations of the study reported in [79] are detailed in Section S6 (Supplementary Materials).

However, the authors of [79] also suggested that differences in the aldehyde profiles between

their two groups may arise from those between the FA compositions of their diets [73], i.e. with a

Nutrients 2020, 12, 974 23 of 49

possible higher peroxidised PUFA and therefore aldehyde content of those received by the CHF one,

but they also indicated that such systematic dietary variations between them were unlikely.

In the control group of participants, the order of decreasing total blood plasma total trans-2-

alkenal concentrations were (peroxidised FA source(s) in brackets, with L, -Ln, -Ln, O, Po and Ar

representing predominant linoleoyl-, -linolenoyl-, -linolenoyl-, oleoyl-, palmitoleoyl- and

arachidonylglycerols respectively) trans-2-octenal (L) > trans-2-hexenal (-Ln - minor aldehydic LOP,

but also a major dietary flavouring agent (section 3.1)) > trans-2-heptenal (O and L) > trans-2-nonenal

(Po, Ar and -Ln, but also a food flavouring agent); that for n-alkanals was n-heptanal (L) > -nonanal

(O) > -octanal (O) > -hexanal (Ar/L, but also derived from the decomposition of trans,trans-2,4-

decadienal [11]); and that for 4-hydroxy-trans-2-alkenals was HHE (-Ln) > HNE (L and Ar) >>> 4-

hydroxy-trans-2-decenal (L) ≈ 4-hydroxy-trans-2-octenal (unknown peroxidised FA sources). As

expected, for the 4-hydroxy-trans-2-alkenals determined, the most predominant ones were those

arising from the sequential peroxidation of α-linolenoyl- (HHE) and linoleoyl-/arachidonoylglycerols

(HNE).

Using the somewhat broad assumption that a highly significant fraction of at least some of these

blood plasma aldehyde levels arise from dietary sources, it should be considered that those found

therein represent only residual concentrations, i.e. what remains following their in vivo consumption

through their metabolic fate in the GI system, in vivo absorption and then further metabolism

thereafter in organs such as the liver, along with any biotransformation of them in human blood

plasma and other environments, e.g. the generation of protein carbonyl species from the reaction of

-unsaturated aldehydes with plasma proteins such as human serum albumin and gamma-

globulins, and additional Schiff base products arising from the reactions of all possible aldehydes

with free primary and secondary amine functions present in selected biomolecules, for example.

‘Between-aldehyde class’ differences in the rates and extents of their consumption will also account

for those observed between their relative blood plasma concentrations. In principle, since -

unsaturated aldehydes are more chemically-reactive than saturated ones [82], should we perhaps

expect higher n-alkanal:trans-2-alkenal ratios in human blood plasma than what is found in fried food

products? Indeed, this ratio is already significantly > 1 in fried potato chip samples (mean ± SEM 1.39

± 0.10 for our dataset (Table 2); 95% confidence intervals 1.18-1.61, p < 0.01) than it is in the culinary

oil sources of these aldehydes, and this has been attributed to their differential levels of reactivity

with potato proteins, amino acids and further biomolecules between these two classes of LOP toxins

[3,14]. This was indeed the case in the above normal LV function group plasma profiles explored, the

ratio being 1.53; however, this difference observed was found not to be statistically significant from

that found in fried potato chips (one sample t-test). Notwithstanding, it may be conjectured that these

proportionately lower control group blood plasma trans-2-alkenal levels may also arise from a higher

level of reactivity of them in vivo. Interestingly, the fractional blood plasma MDA aldehydic LOP

content in these control participants (only 2%) was found to be ca. 3-fold greater than that observed

in potato chip samples (0.60%). Nevertheless, this observation again confirms that MDA represents

only a minor secondary LOP.

Overall, also important is the observation that the total unsaturated aldehyde content of normal

LV function patient blood plasma is significantly greater than that of saturated aldehydes, and this is

indeed also the case for estimated weight percentage human dietary intakes of these toxins by

humans in Refs. [19,85,86], i.e. an unsaturated:saturated aldehyde ratio of 5:2 in mg/kg units [85,86].

Corresponding weight percentage (ppm) values for fried potato chip samples were found to be a very

similar value of ca. 3:1 [12].

A further study performed by Ogihara et. al. in 1999 [87], which determined the blood plasma

concentrations of secondary aldehydic LOPs in premature infants with and without chronic lung

disease (CLD), was, however, limited to only 3 long-chain n-alkanals and 4 trans-2-alkenals, together

with HNE. Full details of this study are available in Section S7 of the Supplementary Materials.

However, it is clear that further research investigations targeted on dietary patterns, human

intake, GI fate, absorption, biodistribution and further metabolism of such dietary LOPs are required

in order to ratify potential relationships between their dietary availabilities and those detected in

Nutrients 2020, 12, 974 24 of 49

human biofluids and tissues. Although not simply conceivable for all patient and age-matched

control groups investigated in human trials, it is also thoroughly recommended that for future clinical

studies focused on explorations of oxidative stress in vivo (particularly the in vivo generation of LOPs

such as reactive aldehydes), all participants involved should be fasted for a sufficient minimal time

period prior to the collection of biofluid or biopsy samples for analysis. Such an approach will

presumably overcome any interferences or confounding effects arising from dietary LOP sources.

4. Atherosclerosis and its Cardiovascular Disease Sequelae

Covalent structural modification of lysyl and further selected amino acid residues of the apo-B

protein composite of low-density-lipoprotein (LDL) by various aldehydes (including trans-2-

alkenals, 4-hydroxy-trans-2-alkenals, acrolein and malondialdehyde, but not exclusively so) affords

its uptake by macrophages to form foam cells, which in turn give rise to artery-blocking fatty streaks.

Indeed, Staprans et. al. [56] discovered that feeding an oxidized lipid-rich diet to New Zealand white

rabbits culminated in a 100% increase in fatty streak lesions within the aorta over those fed an

unoxidized lipid control diet. Interestingly, rabbits receiving the oxidized lipid diet were found to

have a >100% increase in total cholesterol in the pulmonary artery (predominantly as cholesteryl

esters). HNE- and MDA-modified proteins have been previously identified in atherosclerotic lesions

using immunological methods and techniques (reviewed in [88]). Such aldehydes, which can also be

generated in vivo, are also implicated in a range of pathologies arising from or linked to

atherosclerosis, such as their cardiovascular disease sequelae, and complications arising from poorly-

controlled types 1 and 2 diabetes.

Major aldehydic LOPs associated with the chemopathology and pathobiology of atherosclerotic

oxidant injury have included trans-2-alkenals, 4-hydroxy-trans-2-alkenals such as HNE, MDA and 4-

ketoaldehydes [86], although it should be noted that the most predominant ones present in fried food

sources are trans-2-alkenals, trans,trans-2,4-alkadienals and n-alkanals (section 3.1 [3,14]). Moreover,

those arising from dietary sources also include 4,5-epoxy-trans-2-alkenals, cis,trans-alka-2,4-dienals

and hydroperoxy- and hydroxy-trans-2-alkenals, for example. As delineated above, all these

aldehydes readily react with proteins to form relatively stable protein-aldehyde adducts via Schiff

base and Michael addition reactions, and these potential biomarker species may be identified and

determined in in vitro models involving LDL, and in biosamples collected from animals in models of

atherosclerosis, together with those from human patients with potentially enhanced risks, or clinical

signs and symptoms of this disorder. Moreover, some aldehydes have been shown to induce

intracellular oxidative stress assaults, and also activate stress signaling pathways that exert effects on

cellular responses to extracellular stimulating agents [89].

Earlier investigations, which focused on exploring the pathological roles of aldehyde-modified

proteins have demonstrated that LDL-aldehyde adducts have an enhanced recognition by

macrophages, and the uptake of these species is therefore significantly increased in these cells [90].

Moreover, Steinberg et. al. [91,92] first recognized that aldehyde adducts of the apolipoprotein B (Apo

B) component of LDL transforms this lipoprotein to a pro-atherogenic form which is readily taken

up by macrophages to generate foam cells. Further studies which focused on the Apo B moiety of

‘oxidised’ LDL, featured MDA as Nε-(2-propanal)-lysine adducts [93] and 1-amino-3-iminopropene

MDA-lysine cross-links [94]; acrolein adducts, including N-(3-methylpyridinium) lysine [95] and 3-

formyl-3,4-dehydropiperidine species [96,97]; and HNE-derivatized adducts, e.g. enaminal class

HNE-histidine and HNE-lysine species [98].

More recently, Tamamizu-Kato et. al. [99] demonstrated that acrolein markedly impaired the

functional integrity of Apo E, an exchangeable anti-atherogenic apolipoprotein when present at a 10-

fold molar excess, along with heparin-, lipid- and lipid-receptor-binding; experiments were

performed using recombinant Apo E, and immuno-blotting employing an acrolein-lysine-specific

antibody. These studies are fully consistent with the detection of acrolein in atherosclerotic lesions

[96], and acrolein-modified LDL was also found to induce the generation of foam cells from

macrophages [100].

Nutrients 2020, 12, 974 25 of 49

Furthermore, it has been demonstrated that reactive aldehydes suppress mitochondrial

respiration [101], modify ion-channel conductance pathways [102], and diminish myofilament

sensitivity and cardiac contraction [103]. Intriguingly, Wang et. al. [19] found that acrolein effectively

propagates myocardial ischaemic injury and suppresses nitric oxide (NO●)-induced cardioprotection

in mice by a mechanism involving attenuation of protein kinase Cϵ (PKCϵ) signal transduction. In

2011, Ishmail et. al. [104] found that long-term oral exposure to acrolein, at a level consistent with the

human intake of unsaturated aldehydes, gave rise to a dilated cardiomyopathy phenotype in C57BL/6

mice, and from these studies concluded that corresponding effects in humans may be induced by

their exposure to this aldehyde. Therefore, human exposure to environmental/dietary sources of

acrolein and other α,β-unsaturated aldehydes may provide a rational foundation for heart failure.

Since acrolein induces myotube atrophy in vitro, and acrolein-inhalable cigarette smoking serve

as major risk factors for skeletal muscle deterioration (atrophy), a very recent investigation [105]

focused on the mechanism of this phenomenon discovered that low doses of this aldehyde

significantly inhibited myogenic differentiation in vitro, a process which may occur through

suppression of the serine-threonine protein kinase (Akt) signalling pathway. Mice with or without

glycerol-induced muscle injury were exposed to 2.5 and 5 mg/kg BW/day acrolein in distilled water

via the oral route for 4 weeks in order to investigate its effects on muscle wasting and regeneration.

Acrolein’s ability to induce muscle wasting was confirmed in this animal model system, and muscle

regeneration was also found to be retarded. Hence, these data are fully consistent with acrolein’s

potential role in the pathogenesis of myogenesis and disease-linked myopathy. At the cellular level,

exposure to acrolein exerts a wide range of toxic effects, e.g. membrane damage, immune

dysfunction, endoplasmic reticulum stress, and mitochondrial disruption, along with oxidative stress

and protein and DNA adduction [106].

Relationships between the consumption of fried foods in the diet and the risks of cardiovascular

and related diseases have recently been extensively reviewed by Gadariju et. al. [107], to which

readers are referred for further information. This review outlines current evidence available on

associations between human cardiovascular diseases, hypertension, diabetes and obesity, and

estimates of the fried food consumption of population cohorts. However, data acquired in this survey

of many publications focused on this topic were predominantly based on questionnaires to capture

fried food intake information, and the study experimental designs involved were limited to case-

control and cohort investigations. However, on the basis of this (Ref. [107]) review, there is convincing

evidence available to support clear linkages between the risks of these non-communicable chronic

diseases and an increased frequency of fried food consumption, i.e. ≥ 4 times per week.

In the context of metabolic syndrome cluster conditions, further investigations have focused on

the effects of diets containing high contents of oxidised frying oils on the development and/or

progression of type 2 diabetes, and those conducted by Chiang et. al. [67] have demonstrated that

such diets can give rise to lowered levels of insulin secretion and hence glucose intolerance. The

mechanism for these chemopathological impairments appears to involve an oxidative damage-

mediated alteration of glucose metabolism, a process which affects the secretion of insulin by the

pancreatic islets. However, such effects were shown to be circumvented by -TOH supplementation,

an observation supporting the role of this chain-breaking lipid-soluble antioxidant in consuming

highly toxic primary LOO radical species, which in turn suppresses the degradation of any lipid

hydroperoxides formed to biochemically-reactive aldehydes in vivo.

5. Mutagenicity, genotoxicity and carcinogenicity of secondary aldehydic LOPs, and potentially

their dietary/fried food sources

There is now a long and expansive history of research work which has focused on these area for

more than 40 years or so. Indeed, much valuable information is available in this area, with acrolein,

MDA and HNE being the most widely investigated, although there are some limitations with direct

comparative evaluations of these results in view of the wide diversity of cell lines tested and their

tissular sources. Notwithstanding, recent developments in the areas of epigenetic effects, i.e. histon

Nutrients 2020, 12, 974 26 of 49

modification and DNA methylation, are very encouraging e.g. [108]. LOP genotoxicity is extensively

reviewed in [109], and the mutagenicities of carbonyl compounds was fully established in the 1980’s. Moreover, much evidence currently available indicates that aldehydes act as carcinogens [108–

112], or in some cases at least have variable degrees of carcinogenic potential. Moreover, a working

group of the International Agency for Research on Cancer (IARC) found that aldehyde-containing

emissions arising from high temperature frying episodes are “probably carcinogenic to humans

(Group 2A)” [111]. The study performed in [112] has provided a high level of evidence that oxidative

stress mediated by -unsaturated aldehydes significantly contributes towards cytotoxic and

genotoxic cell damage, and these effects are, of course, critically dependent on the structural nature

of the agents tested in this manner. Feron et. al. [113] performed an overall assessment of the cancer

risk status of a range of dietary aldehydes, and from this concluded that although acetaldehyde,

crotonaldehyde and furfural do represent dietary risk factors, this was not the case for acrolein,

formaldehyde, citral and vanillin. However, it was not possible to evaluate this risk factor for MDA,

glycidaldehyde (an acrolein metabolite), benzaldehyde, cinnamaldehyde and anisaldehyde in view

of unavailability of sufficient data. These researchers also concluded that dietary-sourced aldehydes

should be screened for their mutagenic, cytotoxic and cytogenic activities, and emphasised that such

screenings should be prioritised on the basis of their degree of human exposure and expected

mechanisms of action, the latter of which is now a rapidly expanding field of study. Notwithstanding,

this evaluation review [113] is now very dated, and since that time (1991) there have been major

advances in research information and data available on both the dietary availability of these

aldehydes, their in vivo absorption and biodistribution, along with their mechanisms of action and

target organs, in addition to that based on their exertion of deleterious toxicological and carcinogenic

effects.

Attack of endogenous DNA bases by chemically-reactive aldehydic LOPs can represent very

important aetiologies of cancer and human genetic diseases in general. Indeed, such structurally-

modified DNA adducts arising therefrom can give rise to frameshift mutations [114]. MDA

represents a relatively minor aldehydic LOP arising from the peroxidation of -3 FAs containing ≥ 3

carbon-carbon double bonds such as eicosapentaenoic (EPA) and docosahexaenoic acid (DHA)

acylglycerol derivatives (containing five and six double bond units respectively), and which are

prevalent constituents of marine oils.

As an illustration of the pattern of aldehydic LOPS generated from the peroxidation of -3 FAs,

Figure 5 shows two-dimensional (2D) 600 MHz 1H-1H correlation spectroscopy (COSY) NMR profiles

of a commercially-available sample of cod liver oil which was exposed to a prolonged (90 min.)

episode of thermal stressing at a standard frying temperature of 180oC in order to explore its

peroxidative resistivity. Although CLO products are predominantly employed as dietary

supplements and not for frying or cooking purposes, this exposure represents an extreme for the

oxidation of ω-3 FAs and further PUFAs therein during increasing periods of storage and exposure

to atmospheric O2 at ambient temperature, and is also a more analytically-specific approach than

alternative, high temperature-dependent methods for monitoring the oxidative susceptibility of such

oils products since it has the ability to determine the molecular nature and levels of a wide range of

LOPs simultaneously. Data acquired demonstrates the major advantages offered by this technique,

specifically the ability to distinguish between, and electronically integrate resonances arising from a

variety of aldehydes and aldehyde classes, including acrolein and 4,5-epoxy-trans-2-alkenals.

Moreover, this analytical approach was also able to provide much valuable information regarding

the molecular natures of five or more saturated aldehyde classifications, including both long- and

short-chain ones (the latter including -3 FA hydroperoxide-derived propanal).

Nutrients 2020, 12, 974 27 of 49

(a)

(b)

Nutrients 2020, 12, 974 28 of 49

(c)

Figure 5. (a) 5.7–10.2 ppm regions of single-pulse (1D) and two-dimensional (2D) 1H-1H COSY spectra

of a commercial cod liver oil product exposed to a LSSFE for a period of 90 min. at 180 °C, with 1H

chemical shift scales (ppm) on the F1 (ordinate) and F2 (abscissa) axes. (b) Expanded 5.98–6.47 (F1

axis) and 9.36–9.73 ppm (F2 axis) region of the above 1H-1H COSY spectrum, revealing linkages

between the C1-CHO, C2-CH=CH- (and in some cases C3-CH=CH-) resonances of trans-2-alkenals,

trans,trans-2,4-alkadienals, cis,trans-2,4-alkadienals, acrolein and 4,5-epoxy-trans-2-alkenals (1, 2, 5,

Acr and Epox, respectively). A1 represents a connectivity between the = 9.484 and 6.119 ppm

resonances, and is tentatively assigned to a trans-2-alkenal classification with a significantly different

carbon chain length range than that giving rise to the characteristic 9.480 ppm signal. (c) Expanded

2.33–2.94 (F1 axis) and 9.68-9.91 ppm (F2 axis) region of the 1H-1H COSY spectrum shown in (a),

exhibiting clear distinctions between connectivities arising from three long-chain (A, A1 and B) and

one short-chain (D) n-alkanal classification. C represents the 1H-1H correlation for the -CHO and -

CH2 function protons of 4-oxo-n-alkanals. Samples were prepared for 1H-NMR analysis by the

method described in [11], and spectra were acquired on the NMR facility described in Figure 2.

Notwithstanding, such FAs are only present at relatively low levels in most vegetable-based

COs, which are rich in -6 linoleoyl-, but only limited or deplete in -3 linolenoylglycerols, such as

sunflower oil with ≤ 0.20-0.30% (w/w) [12]. However, relatively higher concentrations of -3 FAs are

present in canola and soybean oils, which have approximately 10% and 7% (w/w) of them

respectively. Linseed oil is a notable exception, but this oil is far too dangerous to employ for standard

shallow- or deep-frying episodes in view of its highly explosive nature, although it is still quite

commonly employed for traditional Chinese wok cooking purposes! Fortunately, Belgium and

France have regulations which sensibly limit the amount of this FA for use in frying oils to only 2%

(w/w) [115], as have some other countries such as Chile.

-3 FAs, e.g. -linolenic acid-containing acylglycerols in rapeseed, soybean and linseed oils, and

those containing EPA and DHA in marine oils, are more susceptible to peroxidation than the

linoleoylglycerols predominant in many vegetable oils, and also give rise to a differential pattern of

aldehydic LOPs following peroxidation (e.g. acrolein, 4-oxo-trans-2-alkenals and low-molecular-mass

n-alkanals such as propanal). Therefore, distinction of their 1 and 2D 1H NMR profiles from those of

-6- and -9-rich vegetable COs is a relatively facile process. As expected, 1H-1H COSY linkages for

acrolein were not detectable in spectra acquired on thermally-stressed samples of sunflower oil (data

not shown). Interestingly, 4-hydroxy-trans-2-alkenals were readily 1H NMR-detectable in thermally-

Nutrients 2020, 12, 974 29 of 49

stressed linoleoylglycerol-rich vegetable oils, together with oleoylglycerol-rich palm oil

(predominantly as HNE, with 1H NMR signals located at = 9.59 (d, C1-CHO), 6.82 (dd, C3-CH=CH-

), 6.31 (dd, C2-CH=CH-) and 4.53 ppm (m, C4-CHOH) [12]), but not in heated marine oils, in which

the corresponding aldehyde generated from fragmentation of such FA hydroperoxides is HHE.

Below, the potential or proven mutagenic, genotoxic and carcinogenic actions of MDA, trans-2-

alkenals in general, 4,5-epoxy-trans-2-alkenals, and acetaldehyde and formaldehyde, are reviewed

and discussed, as are those of acrolein, the latter with special reference to the exposure of humans to

wok cooking episodes. Associations cancer risk and fried food intake levels are also described.

Section S5 (Supplementary Materials) provides an outline of the toxicological properties and

potential adverse health effects of HNE, and its lower HHE homologue.

5.1. MDA

Early studies involving an E. coli mutagenesis system revealed that MDA is indeed mutagenic

in cells which feature active DNA-repair systems, and these results indicated that this aldehyde had

the ability to induce inter-strand cross-linking (fluorescent products were detected from such reaction

systems) [116]. Much later, this dialdehyde was found to react with the DNA base adduct guanine to

form the exocyclic adduct, pyrimido(1,2-)purin-10(3H)-one derivative (M1G) [117], and therefore

when MDA is absorbed in vivo [75], it has the ability to generate this derivative. The M1G adduct has

been detected in selected healthy human tissues, including colorectal mucosa [118], and it induces

sequence-dependent frameshift mutations and base pair substitutions in bacteria and in mammalian

cells. This finding suggests a potential role for the M1G lesion in the induction of mutations commonly

related to human diseases. Another early study [119] found that administration of MDA as it enolate

anion sodium salt (throughout a 0.1–10.0 g/g/day dosage level range) to mice in drinking water for

a duration of 12 months caused dose-related hyperplastic and neoplastic alterations to liver nuclei,

but no gross hepatic tumours were generated. However, addition of MDA to the medium of cultured

rat skin fibroblasts gave rise to nuclear abnormalities at added concentrations of only 1.0 µmol/L,

despite a cellular uptake of only 4%. MDA/(3-hydroxy acrolein) have been shown to exert cancer-

initiating activities in female Swiss mice [120,121].

5.2. trans-2-Alkenals

The mutagenicities of 2-hexenal, -heptenal, -octenal and -nonenal have been previously

evaluated in bacterial systems [122], and each of these was found to exert significant effects at µmol/L

concentrations; since 2-hexenal occurs naturally in a range of foods, it has received particular focus

in such investigations. Testing of these aldehydes, along with 2-pentenal, in V79 Chinese hamster

cells at added levels of 3.0–300 µmol/L demonstrated that all of them gave rise to a dose-dependent

enhancement in 6-thioguanine-resistant mutant frequency effect, which increased with increasing

molecular size [123]. Furthermore, 2-nonenal at doses of only 0.10 and 10 µmol/L was found to give

rise to notable sister chromatid exchanges (SCEs), although no chromosomal aberrations, nor

micronuclei, were observed in these studies. A further report, focused on trans-2-hexenal

detoxification and its DNA adduct formation in humans [124], is also featured in Section S8 of the

Supplementary Materials. However, although estimates of the mean daily intake of trans-2-hexenal

for a ‘normal’ diet are 4.2 mg per mean 70 kg BW human, that for a trans-2-hexenal-rich diet is as

much as 42–147 mg/day [125], a range substantially greater than the 12.5 mg 95th percentile intake

estimate of Ref. [124].

In 2005, Nadasi et. al. [125] evaluated the potential carcinogenicity of trans-2-hexenal, for which

humans have a dietary pattern-dependent continuous intake, and for this purpose monitored Ha-ras

and p53 gene expression alterations, together with tumour development in mice and rats following

its administration. For short-term experiments, this study involved CBA/Ca(H-2K), AKR/J(H-2K) and

C3He-mg(H-2K) mice (6–8 weeks old) and Long-Evans, Wistar and Fischer 344 rats (6 females and 6

males of each strain), each receiving 3 x 50 mg/kg BW trans-2-hexenal in a corn oil vehicle orally (age-

matched controls received the same volume of unspiked corn oil). Animals were autopsied 24, 48 and

72 hr. following administration of the aldehyde or its vehicle alone. However, in a long-term study,

Nutrients 2020, 12, 974 30 of 49

mice and rats received 150 mg/kg body weight of trans-2-hexenal in total intraperitoneally (i.p.), i.e.

50 mg/kg on the 1st, 8th and 15th days of the investigation, and were then autopsied following an 18-

month survival period. Any developed tumours were removed and 5-µm formalin-fixed, and

paraffin-embedded sections were routinely stained by haematoxylin/eosin, and then examined by

light microscopy. In the short-term study, no gene alterations were noted 24–72 hr. post-

administration. However, ca. 14% of the 72 mice and rats within the long-term study were found to

develop malignant tumours at the 18-month time-point follow-up evaluation. Therefore, despite

exerting no effects on the expression of both onco- and suppressor genes, this reactive -

unsaturated aldehyde displayed a significant carcinogenic potential, which is potentially explicable

by its epigenetic effects, i.e. they appear to be non-genotoxic carcinogens; in general, consistencies

between the genotoxic and carcinogenic effects of compounds is only ca. 90% [125]. This may serve

to explain the lack of genotoxic risk found for this aldehyde reported in [124].

A further noteworthy point is that since trans-2-hexenal arises from other food sources such as

fruits, especially bananas [125], its overall total daily human intake is expected to be inflated by a

consideration of fried food consumption, which may sometimes exceed more than one fried meal per

day, and also possibly the human consumption of serving portions of fried food sources of this -

unsaturated aldehyde greater than 154 g. However, this agent is not one of the more predominant

ones derived from the peroxidation of UFAs [12].

A study which reported comparisons of the cytotoxic and mutagenic properties of the natural

product 2-cyclohexene-1-one with those of a range of dietary aldehydes is discussed in section S9

(Supplementary Materials).

5.3. Acrolein and Chinese wok cooking

Early investigations demonstrated that both i.p. and intravesicular administration of acrolein to

rats gave rise to anomalous levels of cellular proliferation and hyperplasia of bladder urothelium and

epithelium [126]. Furthermore, a greater incidence of an abnormal DNA (2-deoxyadenosine)-acrolein

adduct has been found in liver [127], oral [128] and bladder cancers [129]. Further information

regarding the carcinogenic potential of acrolein as an inhaled or ingested toxin are outlined in section

S10 (Supplementary Materials)

One epidemiological study reported in 2013 found an elevated incidence of lung cancer in non-

smoking Chinese women who cooked/fried food at very high temperatures using a traditional

Chinese-style wok process, and hence had a high level of exposure to cooking oil fumes arising

therefrom [130]. Supporting laboratory evidence for this hypothesis was provided by the observation

that these women participants excreted significantly higher creatinine (Cn)-normalised

concentrations of acrolein and crotonaldehyde (the latter the next higher trans-2-alkenal homologue

from acrolein) as their mercapturate metabolites. No differences were found between these groups

for corresponding urinary levels of benzene mercapturate. In their conclusion, the authors therefore

recommended that domestic kitchen proprietors/users should act to alleviate human exposure to

toxic and carcinogenic cooking oil fumes generated during traditional wok cooking styles by

ensuring that these areas are sufficiently ventilated at the sites involved. A similar recommendation

should, of course, also apply to all commercial/restaurant cooking sites, albeit to a greater, more

expansive extent. Highly detailed reviews of the molecular mechanisms featured in acrolein toxicity

is provided in [106] and [131]. Information regarding the potential carcinogenic properties of

trans,trans-2,4-decadienal present in linoleoylglycerol-rich cooking oil fumes is available in section

S11 (Supplementary Materials). A related epidemiological study performed in 2002 [132] is related in

the Supplementary Materials (section S10). Notably, wok frying practices are also expected to

markedly promote the oxidative deterioration of PUFAs, since these stir-frying approaches generally

use only small volumes of oil per food portion (say, 10–40 mL), and therefore the oil surface area is

very large, and exposure to atmospheric O2 is maximised. Moreover, trace catalytic transition metal

ions from fried foods will promote both the peroxidation of UFAs, and also the breakdown of CHPDs

and HPMs to aldehyde fragments, etc. Similarly, adventitious trace metal ions such as Cu(II) derived

from the wok metal alloy material itself may provide catalytic sources for these processes.

Nutrients 2020, 12, 974 31 of 49

Intriguingly, when applied dermally to mice and rats, acrolein’s glycidaldehyde metabolite

exerts carcinogenic properties [106,133]. Additionally, acrolein is a major lung cancinogen present in

cigarette smoke [134]. Vinyl chloride, which is structurally related to acrolein, has been identified as

a carcinogen in both animals and humans [135]. In cell culture experiments, acrolein can exert

cytotoxic properties at concentrations of < 0.1 µmol/L [136].

Major health threats posed by aldehydes such as acrolein and crotonaldehyde present in

cigarette smoke are also worthy of much consideration; however, this aspect of aldehyde toxicology

is beyond the scope of this work. Notwithstanding, it is important to note that reliable estimates of

the amounts of ingestible aldehydes available in single, average-sized servings of fried potato chips

are not too dissimilar to those derived from the smoking of a mean daily allocation of 25 cigarettes

[137].

5.4. Crotonaldehyde

In 1986, Chung et. al. [138] orally-administered crotonaldehyde (the next higher α,β-unsaturated

homologue of acrolein), which is mutagenic without metabolic activation [139], to F344 rats in

drinking water at concentrations of either 0.60 or 6.00 mmol/L for a 113-week period, and

histologically evaluated liver tumours in these groups against an untreated age-matched control one.

At the lower dose level, crotonaldehyde was found to induce neoplastic lesions in the liver in 9/27

rats; a further 9 had neoplastic nodules, and 2 had hepatocellular carcinomas. At the higher dose

level, however, this aldehyde gave rise to severe liver damage in 43% of these animals, with the

remaining 57% developing abnormal liver cell foci. Although results acquired also indicated that

crotonaldehyde was a weaker tumorigen than the established carcinogen N-nitrosopyrrolidine

(NPYR), they provided strong evidence for its carcinogenicity. Indeed, the incidence of liver tumours

in rats treated with crotonaldehyde and NPYR at equivalent doses (0.60 mmol/L) was 87 and 33%

respectively. α,β-Unsaturated aldehyde concentrations of 0.60 and 6.00 mmol/L are not at all

dissimilar to those of total trans-2-alkenals found in vegetable-based culinary oils exposed to high-

temperature frying durations [12]. In fact, the higher level is lower than those typically determined

in repeatedly-used frying oils. Much further information focused on the carcinogenicity of aldehydes

may be found in [58] and [140].

5.5. 4,5-Epoxy-trans-2-alkenals

In 2017, the FGE.19 EFSA Panel on Food Contact Materials, Enzymes, Flavourings and

Processing Aids concluded that ‘4,5-epoxydec-2-(trans)-enal (FL-no: 16.071) does raise a safety

concern with respect to genotoxicity and, therefore, it cannot be evaluated according to the

Procedure.’ [141]. As noted in our studies [12], 4,5-Epoxy-trans-2-alkenals represent ca. 10 molar % of

the total α,β-unsaturated aldehyde contents of PUFA-rich corn or sunflower oils when thermally-

stressed according to laboratory-simulated shallow frying episodes at 180 °C.

5.6. Acetaldehyde and formaldehyde

Salaspuro [142] found that aldehyde and alcohol dehydrogenase gene polymorphisms (ALDH2

and ADH respectively) are associated with excessive acetaldehyde exposure, and substantially

increase cancer risk in alcohol drinkers, observations which strongly supports the hypothesis that

this saturated aldehyde represents a local carcinogen in oesophageal and gastric cancers.

Interestingly, acetaldehyde can be classified as a tertiary LOP, since it arises from the deterioration of

isomeric alka-2,4-dienals [143], or 2,3- or 4,5-epoxyaldehydes [144,145] during high temperature

frying episodes. Section S3 (Supplementary Materials) provides information on dietary sources and

estimated dietary intakes of acetaldehyde and formaldehyde, most notably alcoholic beverages for

the former. A further study involved human cells collected from patients with a faulty copy of the

BRCA2 breast cancer gene to investigate mechanisms associated with aldehyde-mediated cancer

induction [146], and the investigators found that formaldehyde exposure leads to the degradation of

cellular BRCA2 protein. In those with one faulty copy of its gene (approximately 1 in 100 humans),

Nutrients 2020, 12, 974 32 of 49

this process reduces this protein’s concentration below that which is deemed sufficient for efficient

DNA repair; this process therefore facilitates the induction of cancer.

The International Agency for Research on Cancer (IARC) has classified formaldehyde, another

known aldehydic LOP, as a human carcinogen [147]. Moreover, in 2011, the National Toxicology

Program, an interagency program of the Department of Health and Human Services also classified

formaldehyde as a known human carcinogen in its 12th Report on Carcinogens [148].

5.7. Impact of fried food intake on cancer risks in humans

Particularly notable are epidemiological studies focused on the impact of fried food intake on

cancer risk. In 2013, Stott-Miller et. al. [149] explored links between the male human consumption of

fried foods and prostate cancer risk, and following a review of dietary intake data from more than

3,000 participants, found that this condition was more prevalent amongst those who frequently

consumed deep-fried foods, particularly French fries, fried chicken, fried fish and doughnuts. These

results provide strong evidence for a relationship between fried food intake and prostate cancer risk;

results were found to be more highly significant for a more aggressive disease status. Additionally, a

meta-analysis of published data [150] found that greater fried food intakes induced an estimated 35%

enhancement of prostate cancer risk.

Knecht et. al. [151] found evidence for a positive association between the human intake of fried

meat and combined breast, endometrium and ovarian cancers in women, i.e. female hormone-related

cancers. Moreover, Bosetti et. al. [152] investigated the role of fried food intake on laryngeal cancer

risk in a case-controlled study focused in Italy and Switzerland (> 500 and 1,200 cases and negative

hospital controls respectively), and discovered a significantly elevated risk for participants who had

high consumption rates of fried potatoes (odds ratio 1.9), meat (1.6), fish (3.1) and eggs (1.9).

Both genotoxic and carcinogenic risks linked to the ingestion of repeatedly-boiled sunflower oil

were investigated by Srivastava et. al. [153], and this study found that its oral administration to Wistar

rats resulted in a dose-dependent induction of aberrant cells and micronuclei; such dosing also

depleted antioxidant enzyme availabilities. Moreover, this treatment also influenced hepatic foci,

along with significant decreases in liver mass.

Woutersen et. al. [154] reviewed both animal model and epidemiologic studies focused on the

effects of dietary fat consumption on the risks of breast, colorectal, pancreatic, and prostate cancers,

and found that its increasing intake exerted a significant influence on prostaglandin and leukotriene

biosynthetic routes, and that these properties represented a universal mechanism for such adverse

effects. These researchers also reported that the 50% lethal dose (LD50) values for acrolein in rabbits

and mice were 7 and 40 mg/kg, i.e. there is a wide ‘between-species’ sensitivity to this α,β-

unsaturated aldehyde.

6. Potential Mechanisms for the Toxicity and Health Effects of Dietary Aldehydes

Saturated aldehydes (both short- and long-chain) act as ‘hard’ electrophiles, exerting their toxic

actions through chemical reactions with the primary and secondary amine functions, for example

that of protein lysyl or histidyl residue side-chains. However, -unsaturated aldehydes and

additional alkenals, along with -oxoaldehydes, act as ‘soft’ electrophiles which preferentially react

with ‘softer’ thiol/thiolate functions within protein, peptide (critically GSH) and free cysteine

residues. In general, chemopathological mechanisms available for the toxicity of LOPs can be defined

as either direct or indirect. Direct mechanisms include the formation of adducts with biomolecules,

e.g. reactions of aldehydes with biochemically-critical proteins and DNA in vivo (i.e. adduction

routes), whereas indirect mechanisms may involve the albeit secondary triggering of mitochondrial,

oxidative, and/or endoplasmic reticulum stress, with special reference to their associations with

human diseases and any target tissues and organs affected.

Recently, Xie et. al. [155] suggested a mechanism for the cytotoxicities of the two different classes

of aldehydes, and these involved protein and/or DNA damage. They stipulated the importance of

DNA repair processes as means for protection against damage provoked by less toxic saturated

aldehydes, but not by the unsaturated ones, and hence surmised that inactivation of cells by the more

Nutrients 2020, 12, 974 33 of 49

toxic latter classes occurs via protein adduct formation. Furthermore, this review suggested that DNA

inter-strand crosslinks, but not DNA-protein crosslinks, nor double-strand DNA breakages, are

critical factors for DNA damage resulting from aldehyde attack. In addition, it appears that aldehyde

cytotoxicity, which is DNA damage-independent, is mediated by the loss of intracellular GSH, which

readily traps -unsaturated aldehydes via Michael addition reactions. However, there are only very

low, sub-micromolar levels of aldehydes available in vivo (i.e. residual concentrations following their

metabolism, or protein/DNA adduct formation), for example those found in blood plasma [79].

Hence, thermodynamically, such diminished concentrations have the ability to only chemically

consume an absolute maximum of their equivalent level of intracellular GSH (for 1:1 aldehyde-GSH

conjugates), which represents only a very small fraction of the large intracellular pools of this

scavenging thiol available in vivo, and which are often much higher millimolar concentrations, e.g.

5.5–6.0 mmol/L in whole human blood, which is almost exclusively intracellular [156]. Therefore, it

is conceivable that such low (micromolar or sub-micromolar concentrations) of aldehydes have the

ability to ‘prime’ cells for a cascade of such damaging events following their uptake.

However, this is certainly not expected to be the case within the GI system, where

[aldehyde]:[thiol/GSH] concentration ratios are expected to be much greater (the fate of aldehydes

therein is outlined in section 3.2). However, aldehyde metabolites such as mercapturate conjugates

of the -unsaturated classes (both with and/or without their aldehydic functions reduced or

oxidised to corresponding alcohol or carboxylate anion, respectively), potentially serve as valuable

biomarkers of human exposure to these dietary toxins in biofluids such as human urine and blood

plasma, as may be protein-conjugated aldehyde adducts in the latter. Indeed, blood plasma

concentrations of both the low- and high-molecular-mass classes of these biotransformation products

are likely to be present at much higher levels than the reactive aldehydic precursors themselves in

such biofluids, which renders their detection and quantification more responsive to some lower

sensitivity bioanalytical techniques. Moreover, mercapturate metabolites are also valuable for the

reliable tracking of these aldehydic LOPs in vivo [117]. Interestingly, conceivably higher level thiol-

unsaturated aldehyde Michael addition products in vivo, may also serve as latent sources of these

reactive and more highly toxic LOPs [157]. It is also anticipated that the bulk of circulating -

unsaturated aldehydes may be covalently bound to the cysteine-34 residue of human serum albumin,

since this protein represents the major source of thiol/thiolate anion in this biofluid, and is present at

a concentration of 30 mg/mL (approximately 0.5 mmol/L, with a near-equivalent thiol concentration).

However, alternative albumin amino acid-aldehyde conjugates have been observed for added

acrolein in in vitro experiments, and these involve the covalent modification of its histidyl and lysyl

residues [158].

A series of proteomic-based investigations have confirmed that unsaturated aldehydes inhibit

functionally-important cellular enzyme activities by specifically targeting active-site cysteinyl

residues therein, or more specifically their thiol/thiolate functions. For example, the impairment of

glutathione S-transferase P1-1 (GSTP1-1) activity via Michael-type adduct generation at its Cys-47

residue by a whole range of -unsaturated aldehydes and ketones (a critical factor for

consideration, since this protein detoxifies xenobiotics through glutathione conjugation processes);

suppression of mitochondrial sirtuin 3 (SIRT3) function by reacting with its Cys-280 residue; and

inhibition of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) activity by acrolein via its ability

to form a Michael addition product at its active-site Cys-152 residue (reviewed in [159]).

Notwithstanding, oxidation of thioredoxin 1 is also considered to represent an important mechanistic

factor for consideration.

Aldehydes readily diffuse across cellular membranes in view of their amphiphilic structures,

and therefore have the capacity to covalently react with and hence modify the structure of

biomolecules located within the cytoplasm and nucleus, for example. Hence, such damage may occur

far from their site of generation if formed in vivo [160], and if produced or available extracellularly,

they have the capacity to interact with adjacent cells located remotely from sites of UFA peroxidation;

in such cases, it appears that plasma membrane proteins serve as the primary targets for aldehyde

attack and adduct formation [161]. Such remote attack may be enabled and/or facilitated by the prior

Nutrients 2020, 12, 974 34 of 49

generation of Michael addition adducts with GSH or other endogenous thiols, which represent latent

sources of these aldehydes [157]. Moreover, both endogenous and exogenous (dietary derived)

aldehydic LOPs react with nuclear proteins, and hence attenuate protein expression via chemical

reactions with transcription factors [162].

Pathologically-significant enzymatic targets for aldehydes critically depend on the cell type

involved, and also on the pattern and distribution of such aldehyde-reactive proteins available, along

with cell-penetrating aldehyde levels. Aldehydic protein adduct generation will undoubtedly have

differential physiological ramifications for different protein targets with differing cellular functions,

along with the precise molecular structure of the aldehyde toxin itself. Moreover, the abilities of such

secondary LOPs to reach these targets will also be critically influenced by the intracellular availability

of low-molecular-mass aldehyde scavengers such as thiols (predominantly GSH and L-cysteine),

other scavenging free amino acids such as L-histidine and L-lysine, and biomolecules with free

primary or secondary amine functions (e.g., the secondary metabolite dimethylamine). Intriguingly,

when present at low (non-toxic) concentrations, selected aldehydes (especially HNE) have the ability

to interfere with signal transduction processes, block cellular proliferation and adhesion, and

promote angiogenesis, differentiation and/or apoptosis in cancer cell lines by exerting an influence

on the modulation of gene expression through the formation of covalently-modified protein and/or

DNA adducts [163,164]. These effects appear to represent an example of an aldehyde-triggered

cellular ‘priming’ process, which requires only very low levels of these induction agents.

Although the biomolecular mechanisms of unsaturated aldehyde toxicity are directly linked to

their abilities to form adducts at functionally-important, regulatory cysteine residues in key enzymes,

and hence abrogate their functions, it appears that the onset of toxicological and associated adverse

health effects are not controlled by the impairment of a single protein. Indeed, much data available

now indicates that such toxins effectively suppress an electrophile-responsive proteome, which

comprises cysteine-directed cell-specific proteins. Such considerations are markedly complicated by

the wide range of aldehydes and aldehyde classes identified in both fried foods and their thermally-

stressed CO sources. In addition to their differential dietary availability and intakes, such proteome

inhibition will be strongly mediated by toxicokinetic parameters, including their GI reactivity and

fate, the rate and extent of their in vivo absorption, subsequent metabolism and biodistribution, etc.,

which, in turn, are determined by their physicochemical characteristics such as electrophilicity and

water solubility, etc. Also important is their ability to access such cysteinyl protein residue targets,

which can be limited for at least some unsaturated aldehydes which have structural steric hindrance

[155,159].

Fortunately, powerful batteries of aldehyde-metabolising enzymes are available for the rapid

consumption and removal of aldehyde toxins in the cardiovascular system [165]. However, gene

polymorphisms, which modify the efficacies and extents of aldehyde removal, may significantly

contribute towards human susceptibilities to exposure to these agents, and allelic variations in the

highly polymorphic glutathione S-transferase P has major catalytic function consequences regarding

its abilities to metabolise aldehydes, e.g. acrolein and crotonaldehyde, effectively. Similarly,

polymorphisms in aldehyde-reducing aldo-keto reductases, and/or aldehyde-oxidising aldehyde

dehydrogenases and cytochrome P450s may also have deleterious health implications. Recent

evidence has indicated that a large number of the genes encoding for aldehyde-metabolising enzymes

are triggered by selected natural plant products, for example diallyl-disulphide and -trisulphide, and

dithiol-2-thione (present in garlic and cruciferous vegetables respectively), and hence these agents

and their plant sources may serve to offer humans protection against dietary aldehydic onslaughts

[165].

6.1. Important considerations for HNE and HNE

In 2019, Sottero et. al. [166] critically reviewed the adverse health effects of secondary aldehydic

LOPs and oxysterols; however, their considerations of the former were limited to HHE and HNE,

which are formed in only relatively low quantities during the thermal-stressing of linolenoyl- and

linoleoylglycerols respectively, in culinary frying oils and consequently fried foods, when expressed

Nutrients 2020, 12, 974 35 of 49

relative to those of the more predominant α,β-unsaturated aldehydes (trans-2-alkenals and

trans,trans-alka-2,4-dienals). Such 4-hydroxy-alkenals readily diffuse from the gut into the blood

circulatory system, as do trans-2-alkenals [73] and many additional aldehyde classes.

Subsequent to the digestion of ω-3 FA-rich foods, significant concentrations of HHE were found

to be generated in both static and dynamic in vitro systems modelling gastric and intestinal digestion

[2–4]. Further evidence for the in vivo absorption of dietary sources of both HHE and HNE has been

provided by Awada et. al. in 2012 [76], who demonstrated that the former aldehyde accumulated in

mouse blood after the feeding of these animals with high fat, ω-3 FA-containing diets for a period of

8 weeks. Similarly, this study revealed that mice which were originally fed with HHE had transient

elevations in its plasma level. Moreover, enterocytes treated with that this LOP increased its

barolateral medium concentrations [76], and taken together, these results provide evidence for its

intestinal absorption. Additionally, radioactivity and radiolabelled HNE metabolites have been

detected in the urine, faeces, intestinal contents and major organs of rats following the oral

administration of tritiated HNE to these animals.

The persistence of α,β-unsaturated aldehydes, including HHE and HNE, following the in vitro

digestion of food sources of them, particularly within the lipidic phases of digestion products, was

confirmed by GC-MS analyses, and this again indicates that they are bioavailable to the GI tract for

absorption [167] (further details on this are available in the Supplementary Materials, Section S5).

However, aldehydes detectable in the blood and tissues of raw fish may also arise from the actions

of lipoxygenases [168].

6.1.1. In vivo generation of HNE/HHE from high and sustained dietary supplementation with ω-3

PUFAs?

In vitro models have been developed in order to evaluate the adverse in vivo generation of HHE

and HNE from dietary ω-3 FA sources, and these effectively mimic the digestion of vegetable cooking

and marine oils [2]. Notably, the in vitro digestion of metmyoglobin-containing fish oil emulsions

gave rise to the production of both HHE and HNE (ca. 2 and 7 µM, respectively) within this matrix

[169]. Moreover, for salmon loin and minced beef of equivalent lipid contents, the formation of both

these aldehydes was determined in such digestive fluids following the gastric and intestinal phases,

and the maximal digestive fluid HNE level observed was ca. 2 µM for both food classes, whereas

intestinal digestion of salmon oil gave rise to a higher concentration of HHE than that observed for

minced beef (3.5 versus 2 µM) [170].

6.1.2. Influence of CO consumption on blood plasma levels of HNE and HHE

Hence, it is of much importance to determine the concentrations of dietary-derived aldehydes

in human peripheral blood following the dietary ingestion of cooking oil acylglycerol PUFAs, both

unheated and those subjected to increasing numbers of high-temperature frying episodes. Indeed,

one interesting study conducted by Calzada et. al. [171] involved calculation of the plasma levels of

healthy adult participants who were supplemented with dietary DHA (200–1,600 mg/day)

throughout a 14-day period. Although no change in HHE concentrations were observed at doses of

200 and 400 mg/day, progressively significant elevations in these values were observed the higher

doses administered (800 and 1,600 mg/day), and these reached 60 and 87 nmol/L respectively.

However, it appears that these researchers did not perform essential quality checks on the

peroxidation status of DHA samples administered in these investigations. Moreover, as noted above,

the TBARS test featured in these studies has major artefactual and interference issues associated with

its use as a means to determine secondary LOP levels, and the heating stage involved in the form of

the test employed in this study was 96 °C for a 60 min. period, which is more than sufficient to

peroxidise PUFAs in analytical samples.

Although it is presumed that such HHE may arise from the in vivo peroxidation of fish oil DHA,

it is, of course, conceivable that this secondary LOP was also present in the samples administered to

the above experimental animals or humans; unfortunately, it certainly appears that the fish oil diets

used for these experiments were not tested for aldehydic and other LOPs prior to their

Nutrients 2020, 12, 974 36 of 49

administration, and therefore HHE may itself have been directly administered along with fish oil

EPA and DHA. HHE is a product of DHA and not EPA peroxidation [172], and this observation is

consistent with that of Nagagawa et. al.’s [173].

HNE is detectable at sub-micromolar levels in human cells, tissues and biofluids, and its ‘free’,

non-adducted concentrations in human plasma is 3–125 nM; such values have been shown to be

markedly enhanced (0.1–1.0 µM) in human disease such as coronary and peripheral artery diseases

[174], and rheumatoid arthritis [175].

However, as noted throughout here, 4-hydroxy-trans-2-alkenal levels present in used

linoleoylglycerol-rich culinary frying oils are always ≤ 10% of their total aldehyde content, and less

so in potato chip samples [12], a likely consequence of the increased reactivity of this and indeed

other classes of α,β-unsaturated aldehydes towards food biomolecules such as proteins, peptides and

amino acids, along with acetal/hemiacetal-forming carbohydrates and alcohols, over those of less

reactive saturated aldehydes.

6.1.3. Haem oxygenase-1 expression and dietary marine oil supplementation: potential beneficial

role of HHE

Since haem oxygenase-1 (HO-1) exerts protective actions against a range of diseases, the role of

ω-3 FAs, which are involved in the induction of its expression both in vitro and in vivo, is of much

interest. Intriguingly, Nagagawa et. al. [173] examined the ability of dietary supplementation of fish

oil on the pattern of FAs and their peroxidation products (specifically HHE and HNE) on HO-1

expression within an extremely wide range of tissues (including liver and kidney) and the blood

plasma of C57BL/6 mice, and found that both HHE concentration and HO-1 expression were

upregulated following institution of this dietary regimen. Such changes were correlated with

corresponding increases in DHA but not EPA levels. Overall, these results were proposed to be

consistent with the hypothesis that DHA-derived HHE actually induces HO-1 expression, and that

this aldehyde may be responsible for the HO-1-mediated protective effects exerted by dietary marine

oils when generated from it in vivo.

6.1.4. Cell signalling by HHE and HNE

Briefly, studies focused on the potential involvements of both HHE and HNE in cellular

signalling processes have indicated their roles in the great majority of signal transduction pathways

[176,177], including redox homeostasis, and mediation of key transcription factor activities, e.g. those

of nuclear factor-κB (NF-κB), nuclear erythroid-related factor (Nrf2) and activator protein 1 (AP-1)

[176–179]. Undoubtedly, their high level of reactivities with thiol(ate) and amine functions to form

Michael addition (both) and Schiff base (the latter only) adducts is of crucial importance here.

6.1.5. Aldehydes as the dominant carcinogens present in cigarette smoke

Finally, a very recent observation of much significance has provided a high level of evidence

that aldehydes represent the dominant carcinogens present in tobacco smoke which give rise to DNA

damage, inhibit DNA repair in tobacco smoke carcinogenesis and also prevent many other tobacco

smoke procarcinogens (including 4-(methylnitrosamine)1-(3-pyridyl)-1-butanone and polyaromatic

hydrocarbons) from becoming DNA-damaging agents [180]. On the basis of these results, the authors

of this paper proposed that toxic aldehydes represent the dominant tobacco smoke carcinogens. As

noted in [12], the aldehyde contents of a typical large size serving of restaurant fried potato chips are

not very dissimilar to those available for inhalation during the smoking of a 20–25 allocation of

tobacco cigarettes [137].

7. Targeted Nutrition and Potential Interventional Routes for Eliminating or Alleviating Health

Risks Associated with Dietary LOP Intake in Humans

Potential strategies for alleviating or circumventing health hazards presented by dietary LOPs

represents a widespread area for careful consideration. These include viable means for the determent

Nutrients 2020, 12, 974 37 of 49

of LOP generation in PUFA-rich frying oils such as their prior supplementation with heat-resistant

lipid-soluble antioxidants, or the removal of these toxins via the treatment of repetitively-used COs

with selected LOP-targeted filtration aid materials. However, a major drawback to this antioxidant

fortification approach is that many studies have provided evidence that naturally-occurring or higher

concentrations of plant-derived chain-breaking antioxidants such as - or γ-TOH, or synthetic ones

such as butylated hydroxytoluene (BHT), are only poorly effective in this context in view of the

extremely high level of repetitive thermally-damaging peroxidative recycling bursts often

encountered during standard high-temperature frying periods that COs are often exposed to [1].

Moreover, at least some of these antioxidants are thermally unstable, and they may also be

significantly volatilised at these temperatures (ca. 180 °C) [9,73]. In view of these findings, the future

availability of more powerful and more thermally-stable antioxidants, including a range of unusual

molecules which are not normally considered to act in this capacity (natural or otherwise), may

indeed develop into a productive area for future development.

The employment of currently-available dimethylpolysiloxane polymers, which are surfactants

and anti-foaming agents which also limit exposure of culinary oil surfaces to atmospheric O2 required

for peroxidation, may also be effective for inhibiting LOP generation during deep-frying episodes;

the future customised design and synthesis of more efficient or composite-function derivatives of

these may therefore promote frying oil safety, along with an extension of the frying reuse periods of

CO products. Additionally, technological approaches available involving methylcellulose or

alternative ‘barrier’ agents, which block the uptake of LOP-loaded used PUFA-rich oils by foods fried

therein [181], may also offer a solution to this critically important public health issue. Indeed, fried

potato chip aldehyde toxin contents are strongly and positively correlated with their total lipid

contents, i.e. they are related to the extent of frying oil uptake in this food LOP source [3].

Interestingly, our research work has also revealed that CO LOPs are predominantly present in the

external batter layer of battered fried foods such as chicken or fish, with little or none detectable in

the food component itself (Figure 3), and so this appears to represent a novel means of protecting

fried foods against LOP uptake and human intake, if only consumers were prepared to remove the

battered covering of such foods prior to eating! Unfortunately, this battered layer tends to serve as a

very palatable, savoury and attractive component of such fried meat and fish food products.

However, a notable and highly plausible prophylactic approach is the dietary supplementation

of human fried food consumers (especially those with poor diets, or a high consumption rate of such

foods) with suitable aldehyde-trapping therapies. For example, the amino acid L-cysteine, which is

equipped with an aldehyde-consuming side-chain thiol/thiolate function [164], and/or suitable chain-

terminating antioxidants, although it should be noted that the latter interventional action will only

serve to potentially terminate the further generation of directly-ingested lipid hydroperoxides in the

GI system, and hence block their degradation to more stable aldehydes and other fragmentation

product toxins. Alternatively, prior fortification of this bioenvironment with relatively high levels of

such ingested antioxidants may effectively impair the peroxidation of ingested UFAs which can be

triggered therein [51,52].

One further possible anti-aldehyde strategy involves the antihypertensive drug hydralazine,

which reacts with acrolein and crotonaldehyde to form stable reaction products, for example (1E)-

acrylaldehyde phthalazin-1-ylhydrazone (E-APH) and (1Z)-acrylaldehyde phthalazin-1-ylhydrazone

(Z-APH) from acrolein, and (1E,2E)-but-2-enal phthalazin-1-ylhydrazone (E-BPH) and (1Z,2E)-but-2-

enal phthalazin-1-ylhydrazone (Z-BPH) from crotonaldehyde [182]. This drug is similarly reactive

towards other 2-alkenals, as is its structural analogue dihydralazine. Hydralazine therefore blocks the

cellular toxicity exerted by acrolein, and other 2-alkenals arising as secondary LOPs, and in 2011 Leung et.

al. [183] found that this treatment (described as an ‘anti-acrolein’ initiative, but not exclusively limited to

trapping only this 2-alkenal) significantly diminished myelin damage and improved behavioural outcome

in an experimental mouse model system of autoimmune encephalomyelitis.

Alternatively, plausible targeted manipulations of human levels and activities of aldehyde-

neutralising enzymes could also serve as a means for combating the deleterious exposures to

aldehyde toxins. Indeed, an improved understanding of the biomolecular mechanisms involved in

Nutrients 2020, 12, 974 38 of 49

the induction or stimulation of such enzymes with, for example, selected sulphur-containing natural

plant products [165], may provide valuable information regarding specific therapeutic targets which,

when activated, may offer an enhanced level of cellular protection against the adverse health effects

of exogenous aldehydes.

Notwithstanding, perhaps the best strategic protective approach is for consumers, together with

restaurant and fast-food outlet proprietors, to simply employ COs with only limited PUFA contents

for frying and cooking purposes. Notwithstanding, the avoidance of fried meals cooked in PUFA-

rich oils is not easily achievable when consumers dine in restaurants or purchase take-out fast food

products (especially if they request such frying oil identity information from restaurant staff). Such a

development will serve as the most palpable, easily instigated, and consumer-controllable approach

for directly avoiding or minimizing aldehyde-mediated adverse health effects. One recent study

demonstrated that a MUFA-rich algal frying oil, which contained ca. 90% oleoylglycerols and only ~

5% (w/w) PUFAs, generated only very low levels of aldehydic toxins when exposed to both actual

and laboratory-simulated frying episodes (deep- and shallow-frying processes respectively) [12].

From all the studies reviewed here, evaluation of the possible health-threatening effects and

disease risks of dietary LOPs realistically remains a dauntingly complex task, since these

considerations should be made with special reference to recommended maximum human daily

intake (MHDI) values for these hazardous agents, i.e. those stipulated by relevant regulatory health

authorities and organizations. However, currently documented values are either very limited to

selected aldehydic LOPs such as acrolein, outdated, or even inconsistent between regulatory bodies,

i.e. very few are available. Similar considerations also apply to outdated or unrealistic estimates for

the MHDI values of such toxins, either from dietary or other sources. One approach employed to

date, however, is the determination of an ‘acrolein-adjusted’ MHDI index, which has been employed

to relate potential values for higher molecular weight 2-alkenals to that available for its lowest

homologue class member, acrolein [3,14]: this value is simply determined by dividing the molecular

mass of acrolein by those of higher 2-alkenals (e.g., linoleoylglycerol hydroperoxide-derived trans-2-

octenal), and then multiplying this fractional ratio by the MHDI value of acrolein itself. Currently,

the authors do not consider this approach to be completely satisfactory for trans- and cis-2-alkenals,

and certainly not so for alternative aldehydic LOP classes such as n-alkanals, for example. Hence, the

future consideration, establishment and ratification of many currently unavailable MHDIs for LOPs

of known molecular identities also represent major demands for action. Consumer concerns

regarding the nutritional and health properties of their foods strongly warrant such requirements

[184].

In view of the above considerations, optimizations of combinations of food processing methods

for eliminating or reducing the content of undesirable LOPs will be facilitated, together with

corresponding assessments of the safety of fried and convenience foods, with special reference to the

ever-changing consumer lifestyles of the global population.

Finally, as a further critically important factor, the multitude of previous investigational

scientific reports available which focus on the possible beneficial health effects of dietary PUFAs

should be thoroughly revisited, particularly with regard to those featuring feeding trials with human

participants, or other related cohort epidemiological or meta-analysis studies. On reflection, it

certainly appears that many of these previously conducted studies may be flawed, since the

researchers involved have predominantly neglected the potentially substantial confounding adverse

health effects associated with the intake of LOPs such as aldehydes, which were undoubtedly present

or even prevalent in the oils or diets originally explored in such investigations.

8. Conclusions

Heating of culinary frying oils at temperatures associated with standard frying practices gives

rise to the generation of very high concentrations of cytotoxic and genotoxic aldehydic LOPs from

thermally-promoted, self-propagating oxygen-fuelled recycling peroxidative assaults occurring

therein. These toxins penetrate into and hence are ‘carried’ by foods fried in such media, and therefore

are available for human ingestion. Since the repeated dietary consumption of such LOPs, especially

Nutrients 2020, 12, 974 39 of 49

the -unsaturated classes, may pose serious and chronic hazards to humans, the development of

strategies for overcoming these threats is of paramount importance. Future clinical feeding trial or

epidemiological investigations focused on explorations of the relationships between the incidence

and/or severity of selected human diseases (such as coronary heart disease, cancer, etc.), and the

frequency and level of dietary LOP ingestion, may therefore serve to decipher and clarify the nature

of such relationships. Similarly, previously available reports that PUFA-laden cooking oils are

‘beneficial’ or ‘safe’ for human consumption after being employed for frying or alternative high

temperature cooking purposes may be erroneous and inaccurate, since they predominantly fail to

monitor or even consider any LOPs therein, nor the major public health threats posed by their human

ingestion.

Following their in vivo ingestion, blockage of the activities and functional status of one or more

intracellular protective enzymes at critical active-site cysteinyl residues appears to represent the most

important mechanism for the cyto- and genotoxicities of unsaturated aldehydes. In general,

aldehydes readily cross cell membranes and enter intracellular environments where they may exert

such damaging actions. An analysis of the fractional concentrations of four classes of aldehydic LOPs

in human blood plasma, a study performed here for the first time (Section 3.2.2), demonstrated that

their mean n-alkanal:trans-2-alkenal ratio was similar to that observed in a fried potato chip dataset,

and this may indicate that such aldehyde classes are at least partially dietary-derived, although there

are, of course, many limitations to this form of evaluation. However, proportionate circulating levels

of 4-hydroxy-trans-2-alkenals (including HNE and HHE) and MDA were found to be significantly

much greater than those present in this commonly-consumed fried food source, and again allowing

for the above limitations, these data suggest that these secondary LOPs arise from in vivo peroxidation

episodes. In principle, secondary aldehydic LOPs ingested by humans have the ability to provoke

further cellular ROS generation in vivo, a phenomenon which, in turn, may stimulate further

aldehydic LOP generation and hence amplify and perpetuate any deleterious health effects inducible.

The World Health Organisation (WHO) has indeed identified concerns with the toxicological

and genotoxic potentials of aldehydes [185]. As an example, in 2002 they reported that there were >

30 epidemiological case-control studies focused on populations exposed to formaldehyde (also a

known LOP [186]), and their cancer incidence [187]. Whilst identifying significant concerns on the

inhalation of aldehydes and linked respiratory tract carcinomas, this report makes little reference to

data available in terms of risks arising from food sources. However, non-respiratory tract cancers

were detected in populations exposed to the inhalation of this aldehyde, i.e. multiple myelomas,

pancreatic, colon and brain cancers, amongst others. Furthermore, the European Union Scientific

Committee on Consumer Safety reported on the ingestion of acetaldehyde and its carcinogenicity,

reproductive toxicity and genotoxicity [187]; the cancer risk status of this LOP is detailed in Section

S3 (Supplementary Materials).

Similarly, in view of a potential role of tt-DDE in human carcinogenesis, and its widespread

occurrence in food products, there remain increasing concerns regarding potential associations

between dienaldehyde exposure and the development of human cancers [188]. These concerns are

now strongly supported by the detection of this aldehyde and other -unsaturated ones in fried

foods and thermally-stressed (used) CO sources of this toxin [12], along with high levels detectable

in fumes generated from linoleoylglycerol-rich cooking oils [189]. Additionally, a large amount of

experimental evidence acquired from animal model system investigations have revealed powerful

associations between reproductive and developmental toxicities and exposure to formaldehyde

(extensively reviewed in Ref. [190]); such experiments have involved a range of exposure routes and

dose levels, in different species.

Notably, since the potential adverse health effects of the low content food process contaminants

acrylamide and MCPD derivatives have attracted much significant attention (both in scientific

publications and the media), why is it that toxic aldehydic LOPs, which are present in fried foods at

much greater concentrations, are not receiving a similar level of consideration? The authors have a

high level of public health concern regarding this issue, not least because it is much more widespread,

i.e. it is shared by many other researchers engaged in this increasingly important research area. Is it

Nutrients 2020, 12, 974 40 of 49

not the right time for health authorities and governmental food standards agencies to warn the public

about these very important health threats?

Critical factors which are most likely to play key roles in determining the nature and level of

dietary LOP intake in humans, e.g., shallow versus deep-frying processes, and particularly their

permeation into fried foods available for human consumption such as potato chips, beef patties,

battered chicken portions, etc. should be further investigated. The availability for human

consumption of high, toxicologically-significant (up to 25 ppm for each class) levels of the

predominant classes of toxic aldehydes in servings of fried foods collected directly from fast-food

retail outlets/restaurants, including ubiquitous, globally-accessible large chain ones, should also be

considered in detail. Moreover, the rigorous establishment of currently unavailable ADIs and MHDIs

for the extensive number of dietary aldehydic LOPs is also a key future prospect. Such requirements

are of much importance in view of consumer stakeholder concerns regarding the nutritional and

health properties, both positive and negative, prospectively offered by contemporary foods and

dietary patterns worldwide.

Supplementary Materials: The following are available online at www.mdpi.com/xxx/s1. Section S1: Epoxy-Fatty

Acid (Epoxy-FA) LOPs: In Vivo Absorption and Toxicities; Section S2: Overview of Dietary Sources of LOPs;

Section S3: Food Sources and Cancer Risks of Acetaldehyde and Formaldehyde; Section S4: Comparisons of the

Dietary Availability and Ingestion of Aldehydic LOPs to those of trans-Fatty Acids, Acrylamide and

Monochloro-Propanediol (MCPD) CO/Fried Food/Lipid Product Toxins; Section S5: In vivo Absorption,

Metabolic Fate, Toxicology, and Adverse Health Effects of HNE and HHE; Section S6: Further Limitations of the

Mak et. al. Study (Ref. [79]); Section S7: Blood Plasma Aldehyde Concentrations in Infants with Chronic Lung

Disease; Section S8: DNA Adduct Formation with and Detoxification of trans-2-Hexenal; Section S9: Cytotoxic

and Mutagenic Potential of the Natural Product 2-Cyclohexene-1-one Evaluated against a Range of Dietary

Aldehydes; Section S10: Acrolein as an Inhaled or Ingested Toxin of Carcinogenic Potential: a Special Case for

Consideration; Section S11: Dietary Sources, Inhalation/Ingestion, Cytotoxicity and Genotoxicity of t,t-DDE

Author Contributions: M. G. had the original concept for this work and all supporting experiments described

therein. B.C.P. performed experiments involving the thermal stressing of CLO products, the time-dependent

collection and analysis of LSSFE samples, sample preparation and 1H-NMR spectral data acquisition, together

with NMR data processing, analysis, and interpretation. B.C.P., J.L. and P.B.W. generated and/or finalised all

manuscript Figures. M.G. contributed towards all these work-tasks, together with the full literature review

involved, the statistical analysis of experimental data, and also preparation and finalisation of the manuscript.

P.B.W., B.C.P. and J.L. all reviewed and edited the manuscript, and B.C.P. also contributed towards the

interpretation and assignment of 1H-NMR spectra. M.G. also fully designed and supervised the experimental

components of the investigations. P.B.W. and M.G. designed and produced the graphical abstract. All authors

contributed towards manuscript preparation and development. All authors have read and agreed to the

published version of the manuscript.

Funding: This research received no external funding.

Acknowledgments: All authors are very grateful to the Dave Wetzel and Jie Zhang of Green Pastures Products

Inc. (NE, USA), and Sally Fallon of the Weston A. Price Foundation (DC, USA) for the provision of culinary and

marine oil samples for NMR analysis and further investigations, and also for stimulating discussions. We also

thank the International Trade Centre, United Nations-WTO (Geneva, Switzerland) for non-financial support.

B.C.P is very grateful to De Montfort University, Leicester, UK for the provision of a fees-waiver PhD scholarship

bursary. J. L. is very grateful to Cancer Research UK (CRUK) for providing funding for her post-doctoral research

fellowship based at DMU.

Conflicts of Interest: None of the authors declare any conflicts of interest.

Ethical Approval: This article does not contain any investigations with human participants or experimental

animals performed by any of the authors.

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